Genetically modified non-human animals and methods of use thereof

ABSTRACT

Genetically modified non-human animals are provided that may be used to model human hematopoietic cell development, function, or disease. The genetically modified non-human animals comprise a nucleic acid encoding human IL-6 operably linked to an IL-6 promoter. In some instances, the genetically modified non-human animal expressing human IL-6 also expresses at least one of human M-CSF, human IL-3, human GM-CSF, human SIRPa or human TPO. In some instances, the genetically modified non-human animal is immunodeficient. In some such instances, the genetically modified non-human animal is engrafted with healthy or diseased human hematopoietic cells. Also provided are methods for using the subject genetically modified non-human animals in modeling human hematopoietic cell development, function, and/or disease, as well as reagents and kits thereof that find use in making the subject genetically modified non-human animals and/or practicing the subject methods.

CROSS REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. § 119 (e), this application claims priority to thefiling date of the U.S. Provisional Patent Application Ser. No.61/722,437 filed Nov. 5, 2012; the full disclosure of which is hereinincorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant number5R01CA156689-04 awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

The aim of biomedical research is to gain a better understanding ofhuman physiology and to use this knowledge to prevent, treat or curehuman diseases. Due to practical and ethical barriers to theexperimentation on human subjects, many studies are conducted on smallanimal models, such as the mouse. Animal models of these human diseasesare therefore needed.

For example, in the United States, around 20,000 patients are annuallynewly diagnosed with multiple myeloma (MM), a mostly incurablemalignancy of antibody-secreting terminally differentiated B cells(Hideshima et al., 2007, Nat Rev Cancer. 7:585-98; Kuehl and Bergsagel,2002, Nat Rev Cancer. 2:175-87). MINI is characterized by theinfiltration of malignant plasma cells in the bone marrow (BM) andclinical manifestations include bone disease, hypercalcemia, cytopenia,renal dysfunction, and peripheral neuropathy (Hideshima et al., 2007,Nat Rev Cancer. 7:585-98; Kuehl and Bergsagel, 2002, Nat Rev Cancer.2:175-87). In most cases, MM is preceded by a premalignant conditioncalled monoclonal gammopathy of undetermined significance (MGUS) thataffects around 3% of persons older than 50 years (Landgren et al., 2009,Blood 113:5412-7). Complex heterogeneous genetic abnormalitiescharacterize MINI cells including changes in the karyotype as well asIgH translocations (Kuehl and Bergsagel, 2002, Nat Rev Cancer. 2:175-87;Zhan et al., 2006, Blood 108:2020-8). Plasma cell clones that areamplified in MGUS are thought to have genetic and phenotypic profilessimilar to myelomatous plasma cells (Chng et al., 2005, Blood106:2156-61; Fonseca et al., 2002, Blood 100:1417-24; Kaufmann et al.,2004, Leukemia. 18:1879-82). While mutations in cyclin D genes have beensuggested to drive development of MM, the potential contributions ofother factors have not been conclusively demonstrated (Bergsagel et al.,2005, Blood 106:296-303). Nonetheless, heritable genetic alterations arenot the sole determinants of the behavior of MM cells. Instead,resistance towards drugs and aberrant biological responses towardscytokines are strongly influenced by interactions with themicroenvironment offering an opportunity to develop novel therapeutics.

Like many other tumors, MM is characterized by heterogeneous cellpopulations strongly interacting with non-malignant stroma cells thatcreate a supportive environment (De Raeve and Vanderkerken, 2005, HistolHistopathol. 20:1227-50; Dhodapkar, 2009, Am J Hematol. 84:395-6). TheBM microenvironment for MM cells consists of a diverse extracellularmatrix (ECM) and of cellular components of both hematopoietic andnon-hematopoietic origin. While the BM provides a protected environmentfor normal hematopoiesis, the interaction of MM cells with ECM proteinsand accessory cells plays a crucial role in MM pathogenesis (De Raeveand Vanderkerken, 2005, Histol Histopathol. 20:1227-50; Dhodapkar, 2009,Am J Hematol. 84:395-6; Hideshima et al., 2007, Nat Rev Cancer.7:585-98). Stroma cells, myeloid cells, osteoclasts, and osteoblastsproduce growth factors such as interleukin 6 (IL-6), B-cell activatingfactor (BAFF), fibroblast growth factor, and stroma cell-derived factor1a that activate signal pathways mediating migration, survival, andgrowth of MM cells. In particular, IL-6 produced by stroma cells,osteoclasts, and myeloid cells seems to be a crucial factor in the earlystages and for pathogenesis of MM (De Raeve and Vanderkerken, 2005,Histol Histopathol. 20:1227-50). Similarly, upon interaction with MINIcells, osteoclasts and dendritic cells produce BAFF and/or aproliferation-inducing ligand (APRIL) providing anti-apoptotic signalsthat also increase drug resistance (De Raeve and Vanderkerken, 2005,Histol Histopathol. 20:1227-50; Kukrej a et al., 2006, J Exp Med.203:1859-65).

The major events in cancer pathogenesis—uncontrolled proliferation,survival and spread of the malignant cells—depend on specificcombinations of supportive cell types and soluble factors present inmicroenvironmental niches. Mouse models play an important role incharacterizing key aspects of the driving forces of malignanttransformation and disease in humans. However, they rarely represent thegenetic complexity and clinicopathologic characteristics of humandisease. While xenotransplantation of human tumors intoimmunocompromised mice has been extensively employed, reliableengraftment has typically been feasible only with highly aggressivetumors or cell lines.

The best models currently available to grow human tumor cells areseverely immunodeficient mice that lack B cells, T cells, and NK cells.In the case of MM, engraftment of primary myeloma cells into these micehas been unsuccessful, but primary myeloma cells are able to engrafthuman fetal bone pieces upon co-transplantation into immunocompromisedmice (Yaccoby et al., 1998, Blood 92:2908-13). In this model MM cellsare found in the human bone, but are not detected in the mouse bone orin the periphery demonstrating high residual xenorejection and a needfor the human BM microenvironment (Yaccoby et al., 1998, Blood92:2908-13; Yaccoby and Epstein, 1999, Blood 94:3576-82). Proving itspotential as in vivo model for MM, it was recently demonstrated thatNOD/Scid/γc^(−/−) mice allow the engraftment of several MM cell lines(Dewan et al., 2004, Cancer Sci. 95:564-8; Miyakawa et al., 2004,Biochem Biophys Res Commun. 313:258-62). However, even those mousemodels with low xenorejection have constricted growth environments byvirtue of a large number of factors that do not cross species barriersbut are essential to support growth and survival of transformed cells(Manz, 2007). In vivo models that allow us to probe the complexpathogenic interplay between the tumor and its environment will beessential to design new drugs and therapies.

Therefore there is an unmet need to develop humanized non-human animalsand methods to reliably grow and study human hematopoietic cells,including primary human hematopoietic tumor cells in mice. The presentinvention addresses these unmet needs in the art.

SUMMARY OF THE INVENTION

Genetically modified non-human animals are provided that may be used tomodel human hematopoietic cell development, function, or disease. Thegenetically modified non-human animals comprise a nucleic acid encodinghuman IL-6 operably linked to an IL-6 promoter. In some instances, thegenetically modified non-human animal expressing human IL-6 alsoexpresses at least one of human M-CSF, human IL-3, human GM-CSF, humanSIRPa or human TPO. In some instances, the genetically modifiednon-human animal is immunodeficient. In some such instances, thegenetically modified non-human animal is engrafted with healthy ordiseased human hematopoietic cells. Also provided are methods for usingthe subject genetically modified non-human animals in modeling humanhematopoietic cell development, function, and/or disease, as well asreagents and kits thereof that find use in making the subjectgenetically modified non-human animals and/or practicing the subjectmethods.

In various aspects of the invention, a genetically modified non-humananimal is provided, the genetically modified non-human animal comprisinga genome comprising a nucleic acid encoding human IL-6 operably linkedto an IL-6 promoter, wherein the animal expresses human IL-6 polypeptideunder the regulatory control of the IL-6 promoter. In some embodiments,the genetically modified non-human animal does not express the animal'snative IL-6.

In some embodiments, the genetically modified non-human animal is arodent. In some embodiments, the non-human animal is a mouse. In somesuch embodiments, the IL-6 promoter to which the nucleic acid encodinghuman IL-6 is operably linked is the mouse IL-6 promoter, and the humanIL-6 gene is operably linked to the mouse IL-6 promoter at the mouseIL-6 locus.

In some embodiments, the genetically modified non-human animal furthercomprises one or more additional nucleic acids selected from a nucleicacid encoding human SIRPa under the control of a SIRPa promoter; anucleic acid encoding human M-CSF operably linked to an M-CSF promoter,wherein the animal expresses human M-CSF; a nucleic acid encoding humanIL-3 operably linked to an IL-3 promoter, wherein the animal expresseshuman IL-3; a nucleic acid encoding human GM-CSF operably linked to aGM-CSF promoter, wherein the animal expresses human GM-CSF; and anucleic acid encoding human TPO operably linked to a TPO promoter,wherein the animal expresses human TPO. In some embodiments, thepromoter is the human promoter for the gene. In other embodiments, thepromoter is the non-human animal promoter for the gene. In someembodiments, the genetically modified non-human animal expresses theanimal's corresponding native protein. In other embodiments, thegenetically modified non-human animal does not express the animal'scorresponding native protein.

In some embodiments, the genetically modified non-human animal isimmunodeficient for the non-human animal immune system. In some suchembodiments, the immunodeficient genetically modified non-human animaldoes not express a recombination activating gene (RAG). In some suchembodiments the immunodeficient genetically modified non-human animaldoes not express the IL2 receptor gamma chain (IL2rg, or “γc”). In somesuch embodiments the immunodeficient genetically modified non-humananimal does not express either a RAG (e.g. RAG1, RAG2) or IL2rg.

In some embodiments, the immunodeficient genetically modified non-humananimal is engrafted with human hematopoietic cells to form a geneticallymodified and engrafted non-human animal. In one embodiment, the humanhematopoietic cells are selected from human umbilical cord blood cells,human fetal liver cells, and cells of a human hematopoietic cell line.In one embodiment, the human hematopoietic cells are CD34+ progenitorcells. In one embodiment, the human hematopoietic cells are cancercells. In certain embodiments, the cancer cells are human multiplemyeloma cells.

In some embodiments, the genetically modified and engrafted animal givesrise to a human cell selected from a CD34+ cell, a hematopoietic stemcell, a hematopoeitic cell, a myeloid precursor cell, a myeloid cell, adendritic cell, a monocyte, a granulocyte, a neutrophil, a mast cell, athymocyte, a T cell, a B cell, a plasma cell, a platelet, and acombination thereof. In one embodiment, the human cell is present at 1month, at 2 months, at 3 months, at 4 months, 5 months, 6 months, 7months, 8 months, 9 months, 10 months, 11 months, or 12 months afterengraftment.

In some embodiments, the genetically modified and engrafted animal givesrise to a human hemato-lymphoid system that comprises humanhematopoietic stem and progenitor cells, human myeloid progenitor cells,human myeloid cells, human dendritic cells, human monocytes, humangranulocytes, human neutrophils, human mast cells, human thymocytes,human T cells, human B cells, human plasma cells, and human platelets.In one embodiment, the human hemato-lymphoid system is present at 4months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11months, or 12 months after engraftment.

In some aspects of the invention, methods are provided for generating anon-human animal engrafted with human hematopoietic cells. In someembodiments, methods are provided for generating an animal model ofhuman immune cell development and function. In certain embodiments,methods are provided for generating an animal model of human B celldevelopment and function.

In some embodiments, the methods comprise transplanting a population ofhuman hematopoietic cells to a genetically modified non-human animalthat is immunodeficient and expresses a nucleic acid encoding human IL-6operably linked to an IL-6 promoter. In some embodiments, the animaldoes not express native IL-6. In some embodiments, the IL-6 promoter isthe non-human animal IL-6 promoter and the human IL-6 gene is operablylinked to the non-human animal IL-6 promoter at the non-human animalIL-6 locus. In some embodiments, the non-human animal is a rodent. Insome such embodiments, the non-human animal is a mouse. As such, in someembodiments, the IL-6 promoter to which the nucleic acid encoding humanIL-6 is operably linked is the mouse IL-6 promoter, and the human IL-6gene is operably linked to the mouse IL-6 promoter at the mouse IL-6locus.

In some embodiments, the transplanted population of hematopoietic cellscomprises CD34+ cells. In some embodiments, the transplanted populationof hematopoietic cells comprises cancer cells. In some embodiments, thetransplanted population of cancer cells comprises multiple myelomacells. In some embodiments, the transplanting comprises intrafemoraland/or intratibial injection.

In some embodiments, the immunodeficient, genetically modified animalexpresses at least one additional human nucleic acid selected from thegroup consisting of a nucleic acid encoding human SIRPa operably linkedto a SIRPa promoter; a nucleic acid encoding human M-CSF operably linkedto a M-CSF promoter; a nucleic acid encoding human IL-3 operably linkedto an IL-3 promoter; a nucleic acid encoding human GM-CSF operablylinked to a GM-CSF promoter; and a nucleic acid encoding human TPOoperably linked to a TPO promoter.

In some aspects of the invention, engrafted, genetically modifiednon-human animals expresses a nucleic acid encoding human IL-6 operablylinked to an IL-6 promoter are provided, these engrafted, non-humananimals having been prepared according to the methods described hereinor as known in the art. In some embodiments, the engrafted, geneticallymodified non-human animal is an animal model of human B cell developmentand differentiation

In various embodiments, methods are provided that encompass the use ofhuman hematopoietic cell-engrafted, genetically modified non-humananimals of the subject disclosure. These methods include, for example,methods for the in vivo evaluation of the growth and differentiation ofhematopoietic and immune cells, methods for the in vivo evaluation ofhuman hematopoiesis, methods for the in vivo evaluation of cancer cells,methods for the in vivo assessment of an immune response, methods forthe in vivo evaluation of vaccines and vaccination regimens, methods forthe use in testing the effect of agents that modulate cancer cell growthor survival, methods for the in vivo evaluation of a treatment ofcancer, and methods for the in vivo production and collection of immunemediators, including human antibodies, and for use in testing the effectof agents that modulate hematopoietic and immune cell function. Forexample, in some embodiments, methods are provided for screening acandidate agent for the ability to treat a hematopoietic cancer. In someembodiments, the method comprises contacting a genetically modifiednon-human animal of the present disclosure that has been engrafted withhuman hematopoietic cancer cells with a candidate agent, and comparingthe viability and/or proliferative rate of human hematopoietic cancercells in the contacted engrafted, genetically modified non-human animalto the human hematopoietic cancer cells in a similarly engrafted,genetically modified non-human animal that is not contacted withcandidate agent, wherein a decrease in the viability and/or rate ofproliferation of the human hematopoietic cancer cells in the contactedengrafted, non-human animal indicates that the candidate agent willtreat a hematopoietic cancer. These and other methods will be apparentto the ordinarily skilled artisan from the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. The patent or application file contains at least onedrawing executed in color. Copies of this patent or patent applicationpublication with color drawing(s) will be provided by the Office uponrequest and payment of the necessary fee. It is emphasized that,according to common practice, the various features of the drawings arenot to-scale. On the contrary, the dimensions of the various featuresare arbitrarily expanded or reduced for clarity. For the purpose ofillustrating the invention, there are shown in the drawings embodimentswhich are presently preferred. It should be understood, however, thatthe invention is not limited to the precise arrangements andinstrumentalities of the embodiments shown in the drawings. Included inthe drawings are the following figures.

FIG. 1 is a set of graphs depicting the results of experimentsdemonstrating the engraftment of INA-6 cells in human IL-6 knock-inmice. Soluble IL-6R levels were measured in mice of the indicatedgenotypes transplanted with 5×10⁶ INA-6 cells intravenously. N indicatesthe number of transplanted mice per group.

FIG. 2, comprising Panels A through F, is a set of images depicting thehistological analysis of femurs after intravenous engraftment of INA-6cells. Rag2^(−/−)Il2rg^(null) Il6^(h/h)hSIRPa+ mice were sacrificedeight weeks after engraftment with 5×10⁶ INA-6 cells intravenously.Femurs were fixed in 10% Formalin and decalcified. 10 μM sections werestained with Toluidine blue or directly analyzed for GFP expressionusing a Leica Confocal microscope.

FIG. 3 is a set of images and a graph depicting the analysis of lungsafter intravenous engraftment of INA-6 cells. Rag2^(−/−)Il2rg^(null)Il6^(h/h)hSIRPa+ mice were sacrificed eight weeks after engraftment with5×10⁶ intravenously injected INA-6 cells. Lung tissue was fixed in 10%Formalin and 10 μM sections were directly analyzed for GFP expressionusing a Leica Confocal microscope. Images (left) depict a 10× (top) and63× (bottom) magnification of sections. The graph (right) depicts human116 gene expression measured in indicated tissues and normalized tomurine hprt expression.

FIG. 4 is a set of graphs depicting the results of experimentsdemonstrating the engraftment of INA-6 cells in human IL-6 knock-inmice. Soluble IL-6R levels were measured in mice of the indicatedgenotypes transplanted with 5×10⁵ INA-6 cells intrafemorally. Each lineindicates an individual mouse.

FIG. 5 is a set of images depicting the histological analysis of femursafter intrafemoral engraftment of INA-6 cells. Rag2^(−/−)Il2rg^(null)Il6^(h/h)hSIRPa+ mice were sacrificed four to six weeks afterengraftment with 5×10⁵ intrafemorally injected INA-6 cells. Femurs werefixed in 10% Formalin and decalcified. 10 μM sections were stained withToluidine blue.

FIG. 6 is a set of images and a graph depicting the results of μCTanalysis of murine femurs after INA-6 transplantation.Rag2^(−/−)Il2rg^(null) Il6^(h/h)hSIRPa+ and control mice were sacrificedfour weeks after engraftment with 5×10⁵ intrafemorally injected INA-6cells. Femurs were fixed in 70% ethanol and analyzed using a murine μCT.Trabecular bone and tissue volumes were quantified to calculate theratio between bone to tissue volume. *: p<0.01 by student t-test.

FIG. 7 is a graph depicting the results of μCT analysis of murine femursafter treatment with anti-myeloma drugs. Rag2^(−/−)Il2re^(null)Il6^(h/h)hSIRPa+ were engrafted with 5×10⁵intrafemorally injected INA-6cells and treated biweekly with Velcade® or Zometa®, respectively. Afterfour weeks, mice were sacrificed and femurs were fixed in 70% ethanolfor μCT analysis. Trabecular bone and tissue volumes were quantified tocalculate the ratio between bone to tissue volume.

FIG. 8 is a set of graphs depicting the results of FACS analysis ofprimary cell engraftment in Rag2^(−/−)Il2rg^(null)hSIRPa⁻Tpo^(h/h)Mcsf^(h/h) Il3/Gmcsf^(h/h) Il6^(h/h) mice. Mice were transplanted with1.5×10⁶ intrafemorally injected CD3-depleted bone marrow cells andsacrificed twelve weeks later. Single-cell suspensions were generatedfrom the injected femur, the collateral leg, and the spleen. Cells werestained for mCD45, hCD19, hCD38, and hCD138. FACS plots show eventsafter gating on mCD45-negative cells. Numbers indicate frequency ofCD38+CD138+ cells

FIG. 9 depicts the results of experiments assessing the percentage ofhuman hematopoietic (hCD45+) cells in blood in engrafted mice determinedby flow cytometry. Horizontal bars indicate the respective meanfrequencies.

FIG. 10 depicts the results of experiments assessing the percentage ofhCD45 of B (CD19+), T (CD3+) and myeloid (CD33+) cells in blood inengrafted mice determined by flow cytometry. Only mice with a hCD45percentage higher than 2% are shown.

FIG. 11, comprising Panels A and B, depicts the results of experimentsassessing percentage (Panel A) and number (Panel B) of human CD45+ cellsin BM, spleen and thymus of 20 week engrafted mice. Bars representaverage±SEM of 4/5 mice for group.

FIG. 12, comprising Panels A and B, depicts the results of FACSexperiments assessing human cells. (Panel A) Drawing showing the gatingstrategy used for separating different B cell populations by flowcytometry. (Panel B) Percentage of different B cell subsets within thehuman CD45+CD19+ cells in 20 week old mice. Bars represent average±SEMof 4/5 mice for group.

FIG. 13, comprising Panels A and B, depicts the results of FACSexperiments assessing human cells. (Panel A) Representative flowcytometric analysis of CDS+ B cells in human fetal liver (FL) and 20week old mice. Numbers in the quadrants indicate percentages of cells.All plots are gated on human CD45+ cells. (Panel B) Percentage of CD5 onhuman B cells in BM and spleen of 20 week engrafted mice. Bars representaverage±SEM of 4/5 mice per group.

FIG. 14 depicts the results of FACS experiments assessing human cells.(Panel A) Representative flow cytometric analysis of CD27+ B cells inhuman fetal liver (FL) and 20 week old mice. Numbers in the quadrantsindicate percentages of cells. All plots are gated on human CD45+ cells.(Panel B) Percentage of CD27 on human B cells in BM and spleen of 20week engrafted mice. Bars represent average±SEM of 4/5 mice per group.

FIG. 15 depicts the results of experiments assessing the total human IgMand IgG levels in plasma samples of 12 (Panel A) and 20 (Panel B) weekold mice. Horizontal bars indicate the respective geometric means. Micewith a PB human engraftment lower than 2% were excluded from theanalysis.

DETAILED DESCRIPTION

Genetically modified non-human animals are provided that may be used tomodel human hematopoietic cell development, function, or disease. Thegenetically modified non-human animals comprise a nucleic acid encodinghuman IL-6 operably linked to an IL-6 promoter. In some embodiments, thegenetically modified non-human animal expressing human IL-6 alsoexpresses at least one of human M-CSF, human IL-3, human GM-CSF, humanSIRPa or human TPO. The invention also relates to methods of generatingand methods of using the genetically modified non-human animalsdescribed herein. In some embodiments, the genetically modifiednon-human animal is a mouse. In some embodiments, the geneticallymodified non-human animal described herein is engrafted with humanhematopoietic cells, including either normal or neoplastic cells, orcombinations thereof. In some embodiments, the genetically modifiednon-human animal described herein is engrafted with human multiplemyeloma (MM) cells. In various embodiments, the human hematopoietic cellengrafted, genetically modified non-human animals of the invention areuseful for the in vivo evaluation of the growth and differentiation ofhematopoietic and immune cells, for the in vivo evaluation of humanhematopoiesis, for the in vivo evaluation of cancer cells, for the invivo assessment of an immune response, for the in vivo evaluation ofvaccines and vaccination regimens, for the use in testing the effect ofagents that modulate cancer cell growth or survival, for the in vivoevaluation of a treatment of cancer, for the in vivo production andcollection of immune mediators, including human antibodies, and for usein testing the effect of agents that modulate hematopoietic and immunecell function.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the compositions and methods as more fully described below.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Such terms are found definedand used in context in various standard references illustrativelyincluding J. Sambrook and D. W. Russell, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press; 4th Ed., 2012; F. M.Ausubel, Ed., Short Protocols in Molecular Biology, Current Protocols;5th Ed., 2002; B. Alberts et al., Molecular Biology of the Cell, 4thEd., Garland, 2002; D. L. Nelson and M. M. Cox, Lehninger Principles ofBiochemistry, 4th Ed., W.H. Freeman & Company, 2004; and Herdewijn, P.(Ed.), Oligonucleotide Synthesis: Methods and Applications, Methods inMolecular Biology, Humana Press, 2004. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

The term “abnormal” when used in the context of organisms, tissues,cells or components thereof, includes those organisms, tissues, cells orcomponents thereof that differ in at least one observable or detectablecharacteristic (e.g., age, treatment, time of day, etc.) from thoseorganisms, tissues, cells or components thereof that display the“normal” (expected) respective characteristic. Characteristics which arenormal or expected for one cell or tissue type, might be abnormal for adifferent cell or tissue type.

The term “antibody,” as used herein, includes an immunoglobulin moleculewhich is able to specifically bind to a specific epitope on an antigen.Antibodies can be intact immunoglobulins derived from natural sources orfrom recombinant sources and can be immunoreactive portions of intactimmunoglobulins. The antibodies in the present invention may exist in avariety of forms including, for example, polyclonal antibodies,monoclonal antibodies, intracellular antibodies (“intrabodies”), Fv, Faband F(ab)2, as well as single chain antibodies (scFv), heavy chainantibodies, such as camelid antibodies, and humanized antibodies (Harlowet al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, NY; Harlow et al., 1989, Antibodies: A LaboratoryManual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

“Constitutive” expression includes a state in which a gene product isproduced in a living cell under most or all physiological conditions ofthe cell.

A “coding region” of a gene includes the nucleotide residues of thecoding strand of the gene and the nucleotides of the non-coding strandof the gene which are homologous with or complementary to, respectively,the coding region of an mRNA molecule which is produced by transcriptionof the gene. A “coding region” of a mRNA molecule also includes thenucleotide residues of the mRNA molecule which are matched with ananti-codon region of a transfer RNA molecule during translation of themRNA molecule or which encode a stop codon. The coding region may thusinclude nucleotide residues comprising codons for amino acid residueswhich are not present in the mature protein encoded by the mRNA molecule(e.g., amino acid residues in a protein export signal sequence).

A “disease” includes a state of health of an animal wherein the animalcannot maintain homeostasis, and wherein if the disease is notameliorated then the animal's health continues to deteriorate.

In contrast, a “disorder” in an animal includes a state of health inwhich the animal is able to maintain homeostasis, but in which theanimal's state of health is less favorable than it would be in theabsence of the disorder. Left untreated, a disorder does not necessarilycause a further decrease in the animal's state of health.

A disease or disorder is “alleviated” if the severity of a symptom ofthe disease or disorder, the frequency with which such a symptom isexperienced by a patient, or both, is reduced.

An “effective amount” or “therapeutically effective amount” of acompound includes that amount of compound which is sufficient to providea beneficial effect to the subject to which the compound isadministered. An “effective amount” of a delivery vehicle includes thatamount sufficient to effectively bind or deliver a compound.

“Encoding” includes the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if, for example, transcription and translation of mRNAcorresponding to that gene produces the protein in a cell or otherbiological system. Both the coding strand, the nucleotide sequence ofwhich is identical to the mRNA sequence and is usually provided insequence listings, and the non-coding strand, used as the template fortranscription of a gene or cDNA, can be referred to as encoding theprotein or other product of that gene or cDNA.

As used herein “endogenous” includes any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” includes any material introducedfrom or produced outside an organism, cell, tissue or system.

The terms “expression construct” and “expression cassette” as usedherein include a double-stranded recombinant DNA molecule containing adesired nucleic acid human coding sequence and containing one or moreregulatory elements necessary or desirable for the expression of theoperably linked coding sequence.

As used herein, the term “fragment,” as applied to a nucleic acid orpolypeptide, includes a subsequence of a larger nucleic acid orpolypeptide. A “fragment” of a nucleic acid can be at least about 15nucleotides in length; for example, at least about 50 nucleotides toabout 100 nucleotides; at least about 100 to about 500 nucleotides, atleast about 500 to about 1000 nucleotides, at least about 1000nucleotides to about 1500 nucleotides; or about 1500 nucleotides toabout 2500 nucleotides; or about 2500 nucleotides (and any integer valuein between). A “fragment” of a polypeptide can be at least about 15nucleotides in length; for example, at least about 50 amino acids toabout 100 amino acids; at least about 100 to about 500 amino acids, atleast about 500 to about 1000 amino acids, at least about 1000 aminoacids to about 1500 amino acids; or about 1500 amino acids to about 2500amino acids; or about 2500 amino acids (and any integer value inbetween).

As used herein, the terms “gene” and “recombinant gene” includes nucleicacid molecules comprising an open reading frame encoding a polypeptide.Such natural allelic variations can typically result in 1-5% variance inthe nucleotide sequence of a given gene. Alternative alleles can beidentified by sequencing the gene of interest in a number of differentindividuals. This can be readily carried out by using hybridizationprobes to identify the same genetic locus in a variety of individuals.Any and all such nucleotide variations and resulting amino acidpolymorphisms or variations that are the result of natural allelicvariation and that do not alter the functional activity are intended tobe within the scope of the invention.

“Homologous” as used herein, includes the subunit sequence similaritybetween two polymeric molecules, e.g. between two nucleic acidmolecules, e.g., two DNA molecules or two RNA molecules, or between twopolypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit, e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions, e.g.if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two compound sequences are homologous then the twosequences are 50% homologous, if 90% of the positions, e.g. 9 of 10, arematched or homologous, the two sequences share 90% homology. By way ofexample, the DNA sequences 5′-ATTGCC-3′ and 5′-TATGGC-3′ share 50%homology.

The terms “human hematopoietic stem and progenitor cells” and “humanHSPC” as used herein, include human self-renewing multipotenthematopoietic stem cells and hematopoietic progenitor cells.

“Inducible” expression includes a state in which a gene product isproduced in a living cell in response to the presence of a signal in thecell.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of a compound, composition, vector,or delivery system of the invention in the kit for effecting alleviationof the various diseases or disorders recited herein. Optionally, oralternately, the instructional material can describe one or more methodsof alleviating the diseases or disorders in a cell or a tissue of amammal. The instructional material of the kit of the invention can, forexample, be affixed to a container which contains the identifiedcompound, composition, vector, or delivery system of the invention or beshipped together with a container which contains the identifiedcompound, composition, vector, or delivery system. Alternatively, theinstructional material can be shipped separately from the container withthe intention that the instructional material and the compound be usedcooperatively by the recipient.

The term “nucleic acid” includes RNA or DNA molecules having more thanone nucleotide in any form including single-stranded, double-stranded,oligonucleotide or polynucleotide. The term “nucleotide sequence”includes the ordering of nucleotides in an oligonucleotide orpolynucleotide in a single-stranded form of nucleic acid.

The term “operably linked” as used herein includes a polynucleotide infunctional relationship with a second polynucleotide, e.g. asingle-stranded or double-stranded nucleic acid moiety comprising thetwo polynucleotides arranged within the nucleic acid moiety in such amanner that at least one of the two polynucleotides is able to exert aphysiological effect by which it is characterized, upon the other. Byway of example, a promoter operably linked to the coding region of agene is able to promote transcription of the coding region. Preferably,when the nucleic acid encoding the desired protein further comprises apromoter/regulatory sequence, the promoter/regulatory sequence ispositioned at the 5′ end of the desired protein coding sequence suchthat it drives expression of the desired protein in a cell. Together,the nucleic acid encoding the desired protein and itspromoter/regulatory sequence comprise a “transgene.”

The term “polynucleotide” as used herein includes a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and include a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term includes both short chains, which also commonly are referred toin the art as peptides, oligopeptides and oligomers, for example, and tolonger chains, of which there are many types. “Polypeptides” include,for example, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof. Theterm “peptide” typically refers to short polypeptides. The term“protein” typically refers to large polypeptides.

The term “progeny” as used herein includes a descendent or offspring andincludes the differentiated or undifferentiated decedent cell derivedfrom a parent cell. In one usage, the term progeny includes a descendentcell which is genetically identical to the parent. In another use, theterm progeny includes a descendent cell which is genetically andphenotypically identical to the parent. In yet another usage, the termprogeny includes a descendent cell that has differentiated from theparent cell.

The term “promoter” as used herein includes a DNA sequence operablylinked to a nucleic acid sequence to be transcribed such as a nucleicacid sequence encoding a desired molecule. A promoter is generallypositioned upstream of a nucleic acid sequence to be transcribed andprovides a site for specific binding by RNA polymerase and othertranscription factors. In specific embodiments, a promoter is generallypositioned upstream of the nucleic acid sequence transcribed to producethe desired molecule, and provides a site for specific binding by RNApolymerase and other transcription factors.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

A “recombinant polypeptide” includes one which is produced uponexpression of a recombinant polynucleotide.

The term “regulatory element” as used herein includes a nucleotidesequence which controls some aspect of the expression of nucleic acidsequences. Exemplary regulatory elements illustratively include anenhancer, an internal ribosome entry site (IRES), an intron; an originof replication, a polyadenylation signal (pA), a promoter, an enhancer,a transcription termination sequence, and an upstream regulatory domain,which contribute to the replication, transcription, post-transcriptionalprocessing of a nucleic acid sequence. Those of ordinary skill in theart are capable of selecting and using these and other regulatoryelements in an expression construct with no more than routineexperimentation. Expression constructs can be generated recombinantly orsynthetically using well-known methodology.

The term “specifically binds,” as used herein with respect to anantibody, includes an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. As another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific.

In some instances, the terms “specific binding” or “specificallybinding”, can be used in reference to the interaction of an antibody, aprotein, or a peptide with a second chemical species, to mean that theinteraction is dependent upon the presence of a particular structure(e.g., an antigenic determinant or epitope) on the chemical species; forexample, an antibody recognizes and binds to a specific proteinstructure rather than to proteins generally. If an antibody is specificfor epitope “A”, the presence of a molecule containing epitope A (orfree, unlabeled A), in a reaction containing labeled “A” and theantibody, will reduce the amount of labeled A bound to the antibody.

The term “synthetic antibody” as used herein includes an antibody whichis generated using recombinant DNA technology, such as, for example, anantibody expressed by a bacteriophage as described herein. The termshould also be construed to mean an antibody which has been generated bythe synthesis of a DNA molecule encoding the antibody and which DNAmolecule expresses an antibody protein, or an amino acid sequencespecifying the antibody, wherein the DNA or amino acid sequence has beenobtained using synthetic DNA or amino acid sequence technology which isavailable and well known in the art.

“Variant” as the term is used herein, includes a nucleic acid sequenceor a peptide sequence that differs in sequence from a reference nucleicacid sequence or peptide sequence respectively, but retains essentialbiological properties of the reference molecule. Changes in the sequenceof a nucleic acid variant may not alter the amino acid sequence of apeptide encoded by the reference nucleic acid, or may result in aminoacid substitutions, additions, deletions, fusions and truncations.Changes in the sequence of peptide variants are typically limited orconservative, so that the sequences of the reference peptide and thevariant are closely similar overall and, in many regions, identical. Avariant and reference peptide can differ in amino acid sequence by oneor more substitutions, additions, deletions in any combination. Avariant of a nucleic acid or peptide can be a naturally occurring suchas an allelic variant, or can be a variant that is not known to occurnaturally. Non-naturally occurring variants of nucleic acids andpeptides may be made by mutagenesis techniques or by direct synthesis.

The term “genetically modified” includes an animal, the germ cells ofwhich comprise an exogenous human nucleic acid or human nucleic acidsequence. By way of non-limiting examples a genetically modified animalcan be a transgenic animal or a knock-in animal, so long as the animalcomprises a human nucleic acid sequence.

As used herein, the term “transgenic animal” includes an animalcomprising an exogenous human nucleic acid sequence integrated into thegenome of the animal.

As used herein, by “knock-in” “knock in” or “knockin” includes a geneticmodification that is targeted to a particular chromosomal locus of thenon-human animal genome and inserts a nucleic acid of interest into thattargeted locus. In some instances, thegenetic modification replaces thegenetic information encoded at the chromosomal locus in the non-humananimal with a different DNA sequence.

Genetically Modified Non-Human Animals

In some aspects of the invention, a genetically modified non-humananimal that expresses human IL-6 is provided. By human IL-6 (hIL6) it ismeant the 184 amino acid protein the sequence for which is described at,e.g, Genbank Accession Nos. NM_000600.3 and NP_000591.1. Human IL-6 is asecreted protein that is produced by, for example, T cells, B cells,monocytes, macrophages, fibroblasts, keratinocytes, endothelial cellsand myeloma cells. IL-6 acts through a cell surface heterodimericreceptor complex comprising a binding subunit (IL-6R) and a signaltransducing subunit (gp130). gp130 is a common component of otherreceptors, such the ones for IL-11, IL-27, LIF, whereas the IL-6R ispredominantly restricted to hepatocytes, monocytes, activated B cells,resting T cells and myeloma cell lines. IL-6 plays a central role inhematopoiesis, in immune responses and in acute phase reactions, havingbeen shown to be an important factor for the final maturation of B cellsinto antibody secreting cells (ASC), especially for the expansion ofplasmablasts during the germinal center reaction in the T-dependent (TD)antibody response. IL-6 is required for T cell proliferation in vitroand for generation of cytotoxic T cells (CTL) in vivo, making them moreresponsive to IL-2.

In some aspects of the invention, the genetically modified non-humananimal that expresses human IL-6 also expresses at least one additionalhuman protein selected from human M-CSF, human IL-3, human GM-CSF, humanTPO, and human SIRPa, or any combination thereof. In other words, thenon-human animal that expresses human IL-6 may express one, two three,four or all five of the human proteins selected from hM-CSF, hIL-3,hGM-CSF, hTPO, and hSIRPa. Genetically modified non-human animals thatexpress hM-CSF, hIL-3, hGM-CSF, hTPO, and/or hSIRPa on which the subjectnon-human animals may be designed or from which the subject non-humananimals may be generated are well known in the art, and are discussed ingreater detail in, for example, US Application No. US 2013/0042330 andRathinam et al. 2011, Blood 118:3119-28, disclosing knock-in mice thatexpress human M-CSF; U.S. Pat. No. 8,541,646 and Willinger et al. 2011,Proc Natl Acad Sci USA, 108:2390-2395, disclosing knock-in mice thatexpress human IL-3 and human GM-CSF; U.S. Pat. No. 8,541,646 andRongvaux et al. 2011, Proc Natl Acad Sci USA, 108:2378-83, disclosingknock-in mice that express human TPO; and PCT Application No. WO2012/040207 and Strowig et al. 2011, Proc Natl Acad Sci USA108(32):13218-13223, disclosing transgenic mice that express humanSirpa; the full disclosures of which are incorporated herein byreference.

In various embodiments, the nucleic acid encoding the human protein isoperatively linked to one or more regulatory sequences in a manner whichallows for transcription of the nucleic acid into mRNA and translationof the mRNA into the human protein. The term “regulatory sequence” isart-recognized and intended to include promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are known to those skilled in the art and aredescribed in 1990, Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. In one embodiment, thehuman nucleic acid is expressed by the native regulatory elements of thehuman nucleic acid. In another embodiment, the human nucleic acid isexpressed by the native regulatory elements of the corresponding nucleicacid of the non-human host animal.

Thus, in some embodiments, the nucleic acid encoding human IL-6 isoperably linked to the non-human animal's IL-6 promoter. In otherembodiments, the nucleic acid encoding human IL-6 is operably linked tothe human IL-6 promoter. As another example, in some embodiments, thenucleic acid encoding human M-CSF is operably linked to the animal'sM-CSF promoter. In other embodiments, the nucleic acid encoding humanM-CSF is operably linked to the human M-CSF promoter. As a thirdexample, in some embodiments, the nucleic acid encoding human IL-3 isoperably linked to the animal's IL-3 promoter. In other embodiments, thenucleic acid encoding human IL-3 is operably linked to the human IL-3promoter. As a fourth example, in some embodiments, the nucleic acidencoding human GM-CSF is operably linked to the animal's GM-CSFpromoter. In other embodiments, the nucleic acid encoding human GM-CSFis operably linked to the human GM-CSF promoter. As a fifth example, insome embodiments, the nucleic acid encoding human TPO is operably linkedto the animal's TPO promoter. In other embodiments, the nucleic acidencoding human TPO is operably linked to the human TPO promoter.

The skilled artisan will understand that the genetically modifiedanimals of the invention include genetically modified animals thatexpress at least one human nucleic acid from a promoter. Nonlimitingexamples of ubiquitously expressed promoters useful in the inventioninclude, but are not limited to, DNA pol II promoter, PGK promoter,ubiquitin promoter, albumin promoter, globin promoter, ovalbuminpromoter, SV40 early promoter, the Rous sarcoma virus (RSV) promoter,retroviral LTR and lentiviral LTR, a beta-actin promoter, a ROSA26promoter, a heat shock protein 70 (Hsp70) promoter, an EF-1 alpha geneencoding elongation factor 1 alpha (EF1) promoter, an eukaryoticinitiation factor 4A (eIF-4A1) promoter, a chloramphenicolacetyltransferase (CAT) promoter and a CMV (cytomegalovirus) promoter.Promoter and enhancer expression systems useful in the invention alsoinclude inducible and/or tissue-specific expression systems.Non-limiting examples of tissue-specific promoters useful in theexpression construct of the compositions and methods of the inventioninclude a promoter of a gene expressed in the hematopoietic system, suchas an IL-6 promoter, a M-CSF promoter, an IL-3 promoter, a GM-CSFpromoter, a SIRPA promoter, a TPO promoter, an IFN-f3 promoter, aWiskott-Aldrich syndrome protein (WASP) promoter, a CD45 (also calledleukocyte common antigen) promoter, a Flt-1 promoter, an endoglin(CD105) promoter and an ICAM-2 (Intracellular Adhesion Molecule 2)promoter. These and other promoters useful in the compositions andmethods of the invention are known in the art as exemplified in Abboudet al. (2003, J. Histochem & Cytochem. 51:941-949), Schorpp et al.(1996, NAR 24:1787-1788), McBurney et al. (1994, Devel. Dynamics,200:278-293) and Majumder et al. (1996, Blood 87:3203-3211). Further tocomprising a promoter, one or more additional regulatory elements, suchas an enhancer element or intron sequence, is included in variousembodiments of the invention. Examples of enhancers useful in thecompositions and methods of the invention include, but are not limitedto, a cytomegalovirus (CMV) early enhancer element and an SV40 enhancerelement. Examples of intron sequences useful in the compositions andmethods of the invention include, but are not limited to, the betaglobin intron or a generic intron. Other additional regulatory elementsuseful in some embodiments of the invention include, but are not limitedto, a transcription termination sequence and an mRNA polyadenylation(pA) sequence.

The skilled artisan will also appreciate that in addition to thenaturally occurring human nucleic acid and amino acid sequences, theterms human nucleic acid and human amino acid encompass variants ofhuman nucleic acid and amino acid sequences as well. As used herein, theterm “variant” defines either an isolated naturally occurring geneticmutant of a human or a recombinantly prepared variation of a human, eachof which contain one or more mutations compared with the correspondingwild-type human. For example, such mutations can be one or more aminoacid substitutions, additions, and/or deletions. The term “variant” alsoincludes non-human orthologues. In some embodiments, a variantpolypeptide of the present invention has at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identity to a wild-type humanpolypeptide.

The percent identity between two sequences is determined usingtechniques as those described elsewhere herein. Mutations can beintroduced using standard molecular biology techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. One of skill inthe art will recognize that one or more amino acid mutations can beintroduced without altering the functional properties of human proteins.

Conservative amino acid substitutions can be made in human proteins toproduce human protein variants. Conservative amino acid substitutionsare art recognized substitutions of one amino acid for another aminoacid having similar characteristics. For example, each amino acid may bedescribed as having one or more of the following characteristics:electropositive, electronegative, aliphatic, aromatic, polar,hydrophobic and hydrophilic. A conservative substitution is asubstitution of one amino acid having a specified structural orfunctional characteristic for another amino acid having the samecharacteristic. Acidic amino acids include aspartate, glutamate; basicamino acids include histidine, lysine, arginine; aliphatic amino acidsinclude isoleucine, leucine and valine; aromatic amino acids includephenylalanine, glycine, tyrosine and tryptophan; polar amino acidsinclude aspartate, glutamate, histidine, lysine, asparagine, glutamine,arginine, serine, threonine and tyrosine; and hydrophobic amino acidsinclude alanine, cysteine, phenylalanine, glycine, isoleucine, leucine,methionine, proline, valine and tryptophan; and conservativesubstitutions include substitution among amino acids within each group.Amino acids may also be described in terms of relative size, alanine,cysteine, aspartate, glycine, asparagine, proline, threonine, serine,valine, all typically considered to be small.

Human variants can include synthetic amino acid analogs, amino acidderivatives and/or non-standard amino acids, illustratively including,without limitation, alpha-aminobutyric acid, citrulline, canavanine,cyanoalanine, diaminobutyric acid, diaminopimelic acid,dihydroxy-phenylalanine, djenkolic acid, homoarginine, hydroxyproline,norleucine, norvaline, 3-phosphoserine, homoserine, 5-hydroxytryptophan,1-methylhistidine, methylhistidine, and ornithine.

Human variants are encoded by nucleic acids having a high degree ofidentity with a nucleic acid encoding a wild-type human. The complementof a nucleic acid encoding a human variant specifically hybridizes witha nucleic acid encoding a wild-type human under high stringencyconditions. Nucleic acids encoding a human variant can be isolated orgenerated recombinantly or synthetically using well-known methodology.

In some embodiments, the genetically modified non-human animal thatexpresses a human nucleic acid sequence also expresses the correspondingnon-human animal nucleic acid sequence. For example, and as described ingreater detail below, in certain embodiments, the human nucleic acidsequence is randomly integrated into the genome of the non-human animal,e.g. such that the animal comprises the exogenous human nucleic acidsequence at a locus other than the non-human animal locus encoding thecorresponding non-human animal protein. In other embodiments, thegenetically modified non-human animal that expresses a human nucleicacid sequence does not express the corresponding non-human animalnucleic acid sequence. For example, and as described in greater detailbelow, in certain embodiments, the nucleic acid encoding the humanprotein is introduced into the animal so as to replace genomic materialencoding the corresponding non-human animal protein, rendering theanimal null for the corresponding non-human animal gene and deficientfor the corresponding non-human animal protein. In other words, thenon-human animal is a “knock-in” for the human gene.

Thus, in some embodiments, the genetically modified non-human animalthat expresses human IL-6 also expresses non-human animal IL-6. In otherembodiments, the genetically modified non-human animal that expresseshuman IL-6 does not express non-human animal IL-6. As a second example,in some embodiments, the genetically modified non-human animal thatexpresses human M-CSF also expresses non-human animal M-CSF. In otherembodiments, the genetically modified non-human animal that expresseshuman M-CSF does not express non-human animal M-CSF. As a third example,in some embodiments, the genetically modified non-human animal thatexpresses human IL-3 also expresses non-human animal IL-3. In otherembodiments, the genetically modified non-human animal that expresseshuman IL-3 does not express non-human animal IL-3. As a fourth example,in some embodiments, the genetically modified non-human animal thatexpresses human GM-CSF also expresses non-human animal GM-CSF. In otherembodiments, the genetically modified non-human animal that expresseshuman GM-CSF does not express non-human animal GM-CSF. As a fifthexample, in some embodiments, the genetically modified non-human animalthat expresses human TPO also expresses non-human animal TPO. In otherembodiments, the genetically modified non-human animal that expresseshuman TPO does not express non-human animal TPO.

In some embodiments, the subject genetically modified animal isimmunodeficient. By “immunodeficient,” it is meant that the non-humananimal is deficient in one or more aspects of its native immune system,e.g the animal is deficient for one or more types of functioning hostimmune cells, e.g. deficient for non-human B cell number and/orfunction, non-human T cell number and/or function, non-human NK cellnumber and/or function, etc.

As one example, the immunodeficient animal may have severe combinedimmune deficiency (SCID). SCID refers to a condition characterized bythe absence of T cells and lack of B cell function. Examples of SCIDinclude: X-linked SCID, which is characterized by gamma chain genemutations or loss of the IL2RG gene and the lymphocyte phenotype T(−)B(+) NK(−); and autosomal recessive SCID characterized by Jak3 genemutations and the lymphocyte phenotype T(−) B(+) NK(−), ADA genemutations and the lymphocyte phenotype T(−) B(−) NK(−), IL-7Ralpha-chain mutations and the lymphocyte phenotype T(−) B(+) NK(+), CD3delta or epsilon mutations and the lymphocyte phenotype T(−) B(+) NK(+),RAG1/RAG2 mutations and the lymphocyte phenotype T(−) B(−) NK(+),Artemis gene mutations and the lymphocyte phenotype T(−) B(−) NK(+),CD45 gene mutations and the lymphocyte phenotype T(−) B(+) NK(+), andPrkdc^(scid) mutations (Bosma et al. (1989, Immunogenetics 29:54-56) andthe lymphocyte phenotype T(−), B(−), lymphopenia, and hypoglobulinemia.As such, in some embodiments, the genetically modified immunodeficientnon-human animal has one or more deficiencies selected from an IL2receptor gamma chain deficiency, an ADA gene mutation, an IL7R mutation,a CD3 mutation, a RAG1 and/or RAG2 mutation, an Artemis mutation, a CD45mutation, and a Prkdc mutation.

The subject genetically modified non-human animal may be any non-humanmammal animal, for example, laboratory animals, domestic animals,livestock, etc., that is genetically modified to comprise human IL-6coding sequence operably linked to an IL-6 promoter, e.g., species suchas murine, rodent, canine, feline, porcine, equine, bovine, ovine,non-human primates, etc.; for example, mice, rats, rabbits, hamsters,guinea pigs, cattle, pigs, sheep, goats, and other transgenic animalspecies, particularly-mammalian species, as known in the art. In certainembodiments, the subject genetically modified animal is a mouse, a rator a rabbit.

In one embodiment, the non-human animal is a mammal. In some suchembodiments, the non-human animal is a small mammal, e.g., of thesuperfamily Dipodoidea or Muroidea. In one embodiment, the geneticallymodified animal is a rodent. In one embodiment, the rodent is selectedfrom a mouse, a rat, and a hamster. In one embodiment, the rodent isselected from the superfamily Muroidea. In one embodiment, thegenetically modified animal is from a family selected from Calomyscidae(e.g., mouse-like hamsters), Cricetidae (e.g., hamster, New World ratsand mice, voles), Muridae (true mice and rats, gerbils, spiny mice,crested rats), Nesomyidae (climbing mice, rock mice, with-tailed rats,Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), andSpalacidae (e.g., mole rates, bamboo rats, and zokors). In a specificembodiment, the genetically modified rodent is selected from a truemouse or rat (family Muridae), a gerbil, a spiny mouse, and a crestedrat. In one embodiment, the genetically modified mouse is from a memberof the family Muridae.

In one embodiment, the subject genetically modified non-human animal isa rat. In one such embodiment, the rat is selected from a Wistar rat, anLEA strain, a Sprague Dawley strain, a Fischer strain, F344, F6, andDark Agouti. In oneanother embodiment, the rat strain is a mix of two ormore strains selected from the group consisting of Wistar, LEA, SpragueDawley, Fischer, F344, F6, and Dark Agouti.

In another embodiment, the subject genetically modified animal is amouse, e.g. a mouse of a C57BL strain (e.g. C57BL/A, C57BL/An,C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ,C57BL/10, C57BL/10ScSn, C57BL/10Cr, C57BL/Ola, etc.); a mouse of the 129strain (e.g. 129P1, 129P2, 129P3,129X1, 129S1 (e.g., 129S1/SV,129S1/SvIm), 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac),129S7, 129S8, 129T1, 129T2); a mouse of the BALB strain; e.g., BALB/c;and the like. See, e.g., Festing et al. (1999) Mammalian Genome 10:836,see also, Auerbach et al (2000) Establishment and Chimera Analysis of129/SvEv- and C57BL/6-Derived Mouse Embryonic Stem Cell Lines). In aspecific embodiment, the genetically modified mouse is a mix of anaforementioned 129 strain and an aforementioned C57BL/6 strain. Inanother specific embodiment, the mouse is a mix of aforementioned 129strains, or a mix of aforementioned BL/6 strains. In a specificembodiment, the 129 strain of the mix is a 129S6 (129/SvEvTac) strain.In yet another embodiment, the mouse is a mix of a BALB strain andanother aforementioned strain.

Thus, for example, in some embodiments, the subject genetically modifiednon-human animal is an immunodeficient mouse deficient in B cell numberand/or function, and/or T cell number and/or function, and/or NK cellnumber and/or function (for example, due to an IL2 receptor gamma chaindeficiency (i.e., γ_(c) ^(−/−)) and/or a RAG deficiency), and having agenome that comprises a human nucleic acid, e.g. a nucleic acid encodinghuman IL-6, hM-CSF, hIL-3, hGM-CSF, hTPO, and/or hSIRPa, operably linkedto its corresponding promoter, e.g. a M-CSF, IL-3, GM-CSF, TPO or SIRPapromoter, respectively, wherein the animal expresses the encoded humanprotein(s).

In certain specific embodiments, the subject genetically modified animalis an immunodeficient mouse comprising a nucleic acid encoding humanIL-6 operably linked to an IL-6 promoter at the mouse IL-6 locus, and anucleic acid encoding human SIRPa operably linked to the human SIRPapromoter randomly integrated into the genome of the non-human animal(i.e., the mouse expresses mouse SIRPa), i.e. an immunodeficient hIL6,hSirpa mouse, e.g. a Rag2^(−/−)IL2rg^(−/−) IL-6^(h/+) hSIRPa⁺ mouse or aRag2^(−/−)IL2rg^(−/−) IL-6^(h/h) hSIRPa⁻ mouse. In some suchembodiments, the mouse further comprises a nucleic acid encoding a humanM-CSF operably linked to an M-CSF promoter, a nucleic acid encodinghuman IL-3 operably linked to an IL-3 promoter, a nucleic acid encodinghuman GM-CSF operably linked to a GM-CSF promoter, and a nucleic acidencoding human TPO operably linked to a TPO promoter, i.e. animmunodeficient hIL-6, hSirpa, hM-CSF, hIL-3, hGM-CSF, hTPO mouse, e.g.a Rag2^(−/−)IL2rg^(−/−) IL-6^(h/+) M-CSF^(h/+) IL-3^(h/+) GM-CSP^(h/+)TPO^(h/+) hSIRPa⁺ mouse, a Rag2^(−/−)IL2rg^(−/−) IL-6^(h/+) M-CSF^(h/h)IL-3^(h/h) GM-CSF^(h/h) TPO^(h/h) hSIRPa¹ mouse.

In certain specific embodiments, the subject genetically modified animalis an immunodeficient mouse comprising a nucleic acid encoding humanIL-6 operably linked to an IL-6 promoter and deficient for mouse IL-6, anucleic acid encoding human SIRPa operably linked to the human SIRPapromoter randomly integrated into the genome of the non-human animal(i.e., the mouse still expresses mouse SIRPa), a nucleic acid encodinghuman M-CSF operably linked to an M-CSF promoter and deficient for mouseM-CSF, a nucleic acid encoding human IL-3 operably linked to an IL-3promoter and deficient for mouse IL-3, a nucleic acid encoding humanGM-CSF operably linked to a GM-CSF promoter and deficient for mouseGM-CSF, and a nucleic acid encoding human TPO operably linked to a TPOpromoter and deficient for mouse TPO, i.e. a Rag2^(−/−) IL2rg^(−/−)IL-6^(h/h) M-CSF^(h/h) IL-3^(h/h) GM-CSF^(h/h) TPO^(h/h), hSIRPa+ mouse.

Methods of Making Genetically Modified Non-Human Animals

The subject genetically modified non-human animals may be generatedusing any convenient method for the generation of genetically modifiedanimals, e.g. as known in the art or as described herein.

For example, a nucleic acid encoding the human protein of interest, e.g.IL-6, hM-CSF, hIL-3, hGM-CSF, hTPO, or hSIRPa, may be incorporated intoa recombinant vector in a form suitable for insertion into the genome ofthe host cell and expression of the human protein in a non-human hostcell. In various embodiments, the recombinant vector includes the one ormore regulatory sequences operatively linked to the nucleic acidencoding the human protein in a manner which allows for transcription ofthe nucleic acid into mRNA and translation of the mRNA into the humanprotein, as described above. It will be understood that the design ofthe vector may depend on such factors as the choice of the host cell tobe transfected, the amount of human protein to be expressed, and/or howthe encoding nucleic acid will integrate into the genome of thenon-human host, e.g. as known in the art.

Any of various methods may then be used to introduce the human nucleicacid sequence into an animal cell to produce a genetically modifiedanimal that expresses the human gene. Such techniques are well-known inthe art and include, but are not limited to, pronuclear microinjectionof oocytes, transformation of embryonic stem cells, homologousrecombination and knock-in techniques. Methods for generatinggenetically modified animals that can be used include, but are notlimited to, those described in Sundberg and Ichiki (2006, GeneticallyEngineered Mice Handbook, CRC Press), Hofker and van Deursen (2002,Genetically modified Mouse Methods and Protocols, Humana Press), Joyner(2000, Gene Targeting: A Practical Approach, Oxford University Press),Turksen (2002, Embryonic stem cells: Methods and Protocols in MethodsMol Biol., Humana Press), Meyer et al. (2010, Proc. Nat. Acad. Sci. USA107:15022-15026), and Gibson (2004, A Primer Of Genome Science 2′ ed.Sunderland, Massachusetts: Sinauer), U.S. Pat. No. 6,586,251, Rathinamet al. (2011, Blood 118:3119-28), Willinger et al., (2011, Proc NatlAcad Sci USA, 108:2390-2395), Rongvaux et al., (2011, Proc Natl Acad SciUSA, 108:2378-83) and Valenzuela et al. (2003, Nat Biot 21:652-659).

For example, the subject genetically modified animals can be created byintroducing the nucleic acid encoding the human protein into an oocyte,e.g., by microinjection, and allowing the oocyte to develop in a femalefoster animal. In preferred embodiments, the construct comprising thehuman nucleic acid sequence is injected into fertilized oocytes.Fertilized oocytes can be collected from superovulated females the dayafter mating and injected with the expression construct. The injectedoocytes are either cultured overnight or transferred directly intooviducts of 0.5-day p.c. pseudopregnant females. Methods forsuperovulation, harvesting of oocytes, expression construct injectionand embryo transfer are known in the art and described in Manipulatingthe Mouse Embryo (2002, A Laboratory Manual, 3rd edition, Cold SpringHarbor Laboratory Press). Offspring can be evaluated for the presence ofthe introduced nucleic acid by DNA analysis (e.g., PCR, Southern blot,DNA sequencing, etc.) or by protein analysis (e.g., ELISA, Western blot,etc.). Such methods typically result in the random integration of theinjected nucleic acid sequence—in this instance, the constructcomprising the nucleic acid encoding the human protein of interest—intothe genome of the oocyte and hence the non-human animal, i.e. at a locusother than the locus in the host animal expressing the correspondingprotein.

As another example, the construct comprising the nucleic acid encodingthe human protein may be transfected into stem cells (ES cells or iPScells) using well-known methods, such as electroporation,calcium-phosphate precipitation, lipofection, etc. The cells can beevaluated for the presence of the introduced nucleic acid by DNAanalysis (e.g., PCR, Southern blot, DNA sequencing, etc.) or by proteinanalysis (e.g., ELISA, Western blot, etc.). Cells determined to haveincorporated the expression construct can then be introduced intopreimplantation embryos. For a detailed description of methods known inthe art useful for the compositions and methods of the invention, seeNagy et al., (2002, Manipulating the Mouse Embryo: A Laboratory Manual,3rd edition, Cold Spring Harbor Laboratory Press), Nagy et al. (1990,Development 110:815-821), U.S. Pat. Nos. 7,576,259, 7,659,442,7,294,754, and Kraus et al. (2010, Genesis 48:394-399). Such methods aretypically used in the targeted integration of the transfected nucleicacid sequence—in this instance, the construct comprising the nucleicacid encoding the human protein of interest—into the genome of the stemcells and hence the non-human animal. Often, such methods result in thereplacement of host genomic material, e.g. genomic material encoding thecorresponding host protein, with the nucleic acid encoding the humanprotein of interest.

A genetically modified founder animals can be used to breed additionalanimals carrying the genetic modification. Genetically modified animalscarrying a nucleic acid encoding the human protein(s) of the presentdisclosure can further be bred to other genetically modified animalscarrying other genetic modifications, or be bred to knockout animals,e.g., a knockout animal that does not express one or more of its genes.

In some embodiments, the genetically modified immunodeficient animalscomprise a genome that includes a nucleic acid encoding a humanpolypeptide operably linked to a promoter, wherein the animal expressesthe encoded human polypeptide. In various embodiments, the geneticallymodified immunodeficient non-human animals comprise a genome thatcomprises an expression cassette that includes a nucleic acid encodingat least one human polypeptide, wherein the nucleic acid is operablylinked to a promoter and a polyadenylation signal and further containsan intron, and wherein the animal expresses the encoded humanpolypeptide.

As discussed above, in some embodiments, the subject geneticallymodified animal is an immunodeficient animal. Genetically modifiednon-human animals that are immunodeficient and comprise one or morehuman cytokines, e.g. IL-6, M-CSF, IL-3, GM-CSF, TPO, and/or SIRPa, maylikewise be generated using any convenient method for the generation ofgenetically modified animals, e.g. as known in the art or as describedherein, e.g. DNA injection of an expression construct into apreimplantation embryo or by use of stem cells, such as embryonic stem(ES) cells or induced pluripotent stem (iPS) cells, for example,comprising a mutant SCID gene allele that, when homozygous, will resultin immunodeficiency, e.g. as described in greater detail above and inthe working examples herein. Mice are then generated with the modifiedoocyte or ES cells using, e.g. methods described herein and known in theart, and mated to produce the immunodeficient mice comprising thedesired genetic modification. As another example, genetically modifiednon-human animals can be generated in a non-immunodeficient background,and crossed to an animal comprising a mutant SCID gene allele that, whenhomozygous, will result in immunodeficiency, and the progeny mated tocreate an immunodeficient animal expressing the at least one humanprotein of interest.

Various embodiments of the invention provide genetically modifiedanimals that include a human nucleic acid in substantially all of theircells, as well as genetically modified animals that include a humannucleic acid in some, but not all their cells. In some instances, e.g.targeted recombination, one copy of the human nucleic acid will beintegrated into the genome of the genetically modified animals. In otherinstances, e.g. random integration, multiple copies, adjacent or distantto one another, of the human nucleic acid may be integrated into thegenome of the genetically modified animals.

Thus, in some embodiments, the subject genetically modified non-humananimal may be an immunodeficient animal comprising a genome thatincludes a nucleic acid encoding a human polypeptide operably linked tothe corresponding non-human animal promoter, wherein the animalexpresses the encoded human polypeptide. In other words, the subjectgenetically modified immunodeficient non-human animal comprises a genomethat comprises an expression cassette that includes a nucleic acidencoding at least one human polypeptide, wherein the nucleic acid isoperably linked to the corresponding non-human promoter and apolyadenylation signal, and wherein the animal expresses the encodedhuman polypeptide.

Utility

The genetically modified non-human animals provided in variousembodiments of the present invention find many uses including, forexample, for use as models of growth and differentiation ofhematopoietic cells, for the in vivo evaluation of human hematopoiesis,for the in vivo evaluation of cancer cells, for in vivo study of animmune response, for in vivo evaluation of vaccines and vaccinationregimens, for the use in testing the effect of agents that modulatecancer cell growth or survival, for the in vivo evaluation of atreatment of cancer, for in vivo production and collection of immunemediators, such as an antibody, and for use in testing the effect ofagents that affect hematopoietic and immune cell function.

Towards this end, in some instances, the subject genetically modifiednon-human animal (a “host”) is engrafted with at least one humanhematopoietic cell. In some embodiments, methods are provided forproducing an animal model for studies of the human hematopoietic system,comprising engrafting human hematopoietic cells into a subjectgenetically modified non-human animal (the “host”). In certainembodiments, methods are provided for engrafting human hematopoieticcells into the genetically modified non-human animal disclosed herein.

In some particular instances, the subject genetically modified non-humananimal is engrafted with at least one human multiple myeloma cell. Insome such embodiments, methods are provided for producing an animalmodel for cancer studies, comprising engrafting human multiple myelomacells into a subject genetically modified non-human animal. In some suchembodiments, the invention is a method of engrafting human multiplemyeloma cells into a subject genetically modified non-human animal. Theengrafted human multiple myeloma cells useful in the compositions andmethods of the invention include any human multiple myeloma cell.

The human hematopoietic cells useful in the engraftment of the subjectgenetically modified non-human animals include any convenient humanhematopoietic cell. Non-limiting examples of human hematopoietic cellsuseful in the invention include, but are not limited to, HSC, HSPC,leukemia initiating cells (LIC), and hematopoietic cells of any lineageat any stage of differentiation, including terminally differentiatedhematopoietic cells of any lineage. In some instances, the humanhematopoietic cell is a primary cell, where “primary cells”, “primarycell lines”, and “primary cultures” are used interchangeably herein toinclude acutely isolated cells, or cell cultures that have been derivedfrom a subject and allowed to grow in vitro for a limited number ofpassages, i.e. splittings, of the culture. For example, primary culturesare cultures that may have been passaged 0 times, 1 time, 2 times, 4times, 5 times, 10 times, or 15 times, but not enough times go throughthe crisis stage. In other embodiments, the human hematopoietic cell isfrom a cell line, that is, the cell is from a culture that isimmortalized, e.g. it has been passaged more than about 15 times. Insome instances, the hematopoietic cells that are engrafted comprisehealthy cells. In other instances, the hematopoietic cells that areengrafted comprise diseased hematopoietic cells, e.g. canceroushematopoietic cells, e.g. cancerous effector B cells, i.e. multiplemyeloma cells. In some instances, the hematopoietic cells that areengrafted comprise both healthy and diseased cells, e.g. healthy B cellsand cancerous effector B cells, healthy T cells and cancerous effector Bcells, etc.

Hematopoietic cells, i.e. primary cells, cell lines generated therefrom,etc., can be derived from any tissue or location of a human donor,including, but not limited to, bone marrow, peripheral blood, liver,fetal liver, or umbilical cord blood. Such hematopoietic cells can beisolated from any human donor, including healthy donors, as well asdonors with disease, such as cancer, including leukemia. Engraftment ofhematopoietic cells in the subject genetically modified animal ischaracterized by the presence of human hematopoietic cells in theengrafted animal. In particular embodiments, engraftment ofhematopoietic cells in the subject genetically modified animal ischaracterized by the presence of differentiated human hematopoieticcells in the engrafted animal in which hematopoietic cells are provided,as compared with appropriate control animals.

Isolation of human hematopoietic cells, administration of the humanhematopoietic cells to a host animal and methods for assessingengraftment thereof are well-known in the art. Hematopoietic cells,including either normal and neoplastic cells, or combinations thereof,for administration to a host animal can be obtained from any tissuecontaining hematopoietic cells such as, but not limited to, umbilicalcord blood, bone marrow, peripheral blood, cytokine orchemotherapy-mobilized peripheral blood and fetal liver. Exemplarymethods of isolating human hematopoietic cells, of administering humanhematopoietic cells to a host animal, and of assessing engraftment ofthe human hematopoietic cells in the host animal are described hereinand in Pearson et al. (2008, Curr. Protoc. Immunol. 81:1-15), Ito et al.(2002, Blood 100:3175-3182), Traggiai et al. (2004, Science304:104-107), Ishikawa et al. (2005, Blood 106:1565-1573), Shultz et al.(2005, J. Immunol. 174:6477-6489) and Holyoake et al. (1999, ExpHematol. 27:1418-27).

In some embodiments of the invention, the human hematopoietic cells,including either normal and neoplastic cells, or combinations thereof,are isolated from an original source material to obtain a population ofcells enriched for a particular hematopoietic cell population (e.g.,HSCs, HSPCs, LICs, CD34+, CD34−, lineage specific marker, cancer cellmarker, etc.). The isolated hematopoietic cells may or may not be a purepopulation. In one embodiment, hematopoietic cells useful in thecompositions and methods of the invention are depleted of cells having aparticular marker. In another embodiment, hematopoietic cells useful inthe compositions and methods of the invention are enriched by selectionfor a marker. In some embodiments, hematopoietic cells useful in thecompositions and methods of the invention are a population of cells inwhich the selected cells constitute about 1-100% of the cells, althoughin certain embodiments, a population of cells in which the selectedcells constitute fewer than 1% of total cells can also be used. In oneembodiment, hematopoietic cells useful in the compositions and methodsof the invention are depleted of cells having a particular marker, suchas CD34. In another embodiment, hematopoietic cells useful in thecompositions and methods of the invention are enriched by selection fora marker, such as CD34. In some embodiments, hematopoietic cells usefulin the compositions and methods of the invention are a population ofcells in which CD34+ cells constitute about 1-100% of the cells,although in certain embodiments, a population of cells in which CD34+cells constitute fewer than 1% of total cells can also be used. Incertain embodiments, the hematopoietic cells useful in the compositionsand methods of the invention are a T cell-depleted population of cellsin which CD34+ cells make up about 1-3% of total cells, alineage-depleted population of cells in which CD34+ cells make up about50% of total cells, or a CD34+ positive selected population of cells inwhich CD34+ cells make up about 90% of total cells.

The number of hematopoietic cells administered is not consideredlimiting with regard to the generation of a human hematopoietic and/orimmune system in a genetically modified non-human animal expressing atleast one human gene. Thus, by way of non-limiting example, the numberof hematopoietic cells administered can range from about 1×10³ to about1×10⁷, although in various embodiments, more or fewer can also be used.By way of another non-limiting example, the number of HSPCs administeredcan range from about 3×10³ to about 1×10⁶ CD34+ cells when the recipientis a mouse, although in various embodiments, more or fewer can also beused. For other species of recipient, the number of cells that need tobe administered can be determined using only routine experimentation.

For example, in one embodiment, the genetically modified and treatedmouse is engrafted with human hematopoietic cells or human hematopoieticstem cells (HPSCs) to form a genetically modified and engrafted mouse.In one embodiment, the hematopoietic cells are selected from humanumbilical cord blood cells and human fetal liver cells. In oneembodiment, engraftment is with about 1-2×10⁵ human CD34+ cells.

In some instances, administration of the hematopoietic cells (e.g.,normal or neoplastic) may be preceded by conditioning, e.g. eithersub-lethal irradiation of the recipient animal with high frequencyelectromagnetic radiation, generally using gamma or X-ray radiation, ortreatment with a radiomimetic drug such as busulfan or nitrogen mustard.Conditioning is believed to reduce numbers of host hematopoietic cells,create appropriate microenvironmental factors for engraftment of humanhematopoietic cells, and/or create microenvironmental niches forengraftment of human hematopoietic cells. Standard methods forconditioning are known in the art, such as described herein and in J.Hayakawa et al, 2009, Stem Cells, 27(1):175-182. In one embodiment, thegenetically modified mouse is treated so as to eliminate endogenoushematopoietic cells that may exist in the mouse. In one embodiment, thetreatment comprises irradiating the genetically modified mouse. In aspecific embodiment, newborn genetically modified mouse pups areirradated sublethally. In a specific embodiment, newborn pups areirradiated 2×200 cGy with a four hour interval.

Hematopoietic cells (e.g., normal or neoplastic) can be administeredinto newborn or adult animals by administration via various routes, suchas, but not limited to, intravenous, intrahepatic, intraperitoneal,intrafemoral and/or intratibial. Methods for engraftment of humanhematopoietic cells, including either normal and neoplastic cells, orcombinations thereof, in immunodeficient animals are provided accordingto embodiments of the present invention which include providing humanhematopoietic cells to the immunodeficient animals, with or withoutirradiating the animals prior to administration of the hematopoieticcells. Methods for engraftment of human hematopoietic cells inimmunodeficient animals are provided according to embodiments of thepresent invention which include providing human hematopoietic cells,including either normal and neoplastic cells, or combinations thereof,to the genetically modified non-human animals of the invention, with orwithout, administering a radiomimetic drug, such as busulfan or nitrogenmustard, to the animals prior to administration of the hematopoieticcells.

Engraftment of human hematopoietic cells, including either normal andneoplastic cells, or combinations thereof, in the genetically modifiedanimal of the invention can be assessed by any of various methods, suchas, but not limited to, flow cytometric analysis of cells in the animalsto which the human hematopoietic cells are administered at one or moretime points following the administration of hematopoietic cells.

Generally, engraftment can be considered successful when the number (orpercentage) of human hematopoietic cells, including either normal andneoplastic cells, or combinations thereof, present in the geneticallymodified non-human animal is greater than the number (or percentage) ofhuman cells that were administered to the non-human animal, at a pointin time beyond the lifespan of the administered human hematopoieticcells. Detection of the progeny of the administered hematopoietic cellscan be achieved by detection of human DNA in the recipient animal, forexample, or by detection of intact human hematopoietic cells, such as bythe detection of the human cell marker, such as human CD45, human CD34,or sIL-6R for example. Serial transfer of human hematopoietic cells froma first recipient into a secondary recipient, and engraftment of humanhematopoietic cells in the second recipient, is a further optional testof engraftment in the primary recipient. Engraftment can be detected byflow cytometry as 0.05% or greater human CD45+ cells in the blood,spleen or bone marrow at 1-4 months after administration of the humanhematopoietic cells. A cytokine (e.g., GM-CSF) can be used to mobilizestem cells, for example, as described in Watanabe (1997, Bone MarrowTransplantation 19:1175-1181).

In one embodiment, the immunodeficient genetically modified andengrafted animal gives rise to a human cell selected from a CD34+ cell,a hematopoietic stem cell, a hematopoeitic cell, a myeloid precursorcell, a myeloid cell, a dendritic cell, a monocyte, a granulocyte, aneutrophil, a mast cell, a thymocyte, a T cell, a B cell, a platelet,and a combination thereof. In one embodiment, the human cell is presentat 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10months, 11 months, or 12 months after engraftment.

In one embodiment, the immunodeficient genetically modified andengrafted animal gives rise to a human hemato-lymphoid system thatcomprises human hematopoietic stem and progenitor cells, human myeloidprogenitor cells, human myeloid cells, human dendritic cells, humanmonocytes, human granulocytes, human neutrophils, human mast cells,human thymocytes, human T cells, human B cells, and human platelets. Inone embodiment, the human hemato-lymphoid system is present at 4 months,5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months,or 12 months after engraftment.

In one embodiment, the immunodeficient genetically modified andengrafted animal gives rise to a human hemato-lymphoid system thatcomprises cancerous human hematopoietic cells, for example neoplasticplasma (effector B) cells. In one embodiment, the cancerous humanhematopoietic cells are present at 4 weeks, at 6 weeks, at 8 weeks, at12 weeks, or at more than 12 weeks after engraftment. In certainembodiments, the cancerous human hematopoietic cells are present at 2weeks, at 4 weeks, at 6 weeks, at 8 weeks, at 12 weeks, or at more than12 weeks after engraftment.

Once engrafted with human hematopoietic cells, the subject geneticallymodified non-human animals find many uses in the art. For example,engrafted genetically modified animals of the present disclosure areuseful for studying the function of human hematopoietic cells inperipheral blood. As demonstrated in working example 2, geneticallymodified mice that are immunodeficient and comprise a nucleic acidencoding human IL-6 operably linked to an IL-6 promoter at the IL-6mouse locus (e.g., Rag2^(−/−)IL2rg^(null)IL-6^(h/h) mice,Rag2^(−/−)IL2rg^(null)IL-6^(h/h) hSIRPa¹ mice, and Rag2^(−/−)IL2rg^(−/−)IL-6^(h/h) M-CSF^(h/h) IL-3^(h/h) GM-CSF^(h/h) TPO^(h/h), hSIRPa+)support engraftment of human hematopoietic cells, e.g. CD34⁺ progenitorcells, into the peripheral blood and spleen better than immunodeficientmice that do not express human IL-6, i.e. Rag2^(−/−)IL2rg^(null) mice.Moreover, these genetically modified mice promote the differentiation ofhuman hematopoietic cells more efficiently than immunodeficient micethat do not express human IL-6. For example, these genetically modifiedmice better promote the differentiation of CD5+ B cells and CD27+ Bcells. CD5 is a protein found on a subset of IgM-secreting B cellscalled B-1 cells, and serves to mitigate activating signals from the Bcell receptor so that the B-1 cells can only be activated by very strongstimuli (such as bacterial proteins) and not by normal tissue proteins.CD27 is a marker for memory B cells. Additionally, these geneticallymodified mice support the development of better-functioning humanhematopoietic cells than immunodeficient mice that do not express humanIL-6. For example, B cells differentiate into IgG secreting plasma cellsmore rapidly in these genetically modified mice than in immunodeficientmice that do not express human IL-6. As such, engrafted geneticallymodified animals of the present disclosure find use in studyinghematopoietic cell development and function, and more particularly, Blymphocyte differentiation and function.

As another example, engrafted genetically modified animals of thepresent disclosure are useful for studying hematopoietic cancers. Asdemonstrated in working example 1 below, genetically modified mice thatare immunodeficient and comprise a nucleic acid encoding human IL-6operably linked to an IL-6 promoter at the mouse IL-6 locus, e.g.Rag2^(−/−)IL2rg^(null)IL-6^(h/h) mice, Rag2^(−/−)IL2rg^(null)IL-6^(h/h)hSIRPa⁺ mice, and Rag2^(−/−) IL2rg^(−/−) IL-6^(h/h) M-CSF^(h/h)IL-3^(h/h) GM-CSF^(h/h) TPO^(h/h), hSIRPa+, engraft with primary humanmultiple myeloma cells and cells of human multiple myeloma cell lines,whereas immunodeficient mice that do not express human IL-6, i.e.Rag2^(−/−)IL2rg^(null) mice, do not. Expression of human SIRPa by thegenetically modified host further improves the rate and extent ofengraftment observed. Furthermore, engraftment of the multiple myelomacells directly to bone of these immunodeficient, genetically modifiedmice disclosed herein reproduces the bone pathology typically associatedwith human multiple myeloma, e.g. bone destruction and resorption, e.g.as quantified by μCT scan.

As such, engrafted genetically modified animals of the presentdisclosure find use in screening candidate agents to identify those thatwill treat hematopoietic cancers. The terms “treatment”, “treating” andthe like are used herein to generally include obtaining a desiredpharmacologic and/or physiologic effect. The effect may be prophylacticin terms of completely or partially preventing a disease or symptomthereof and/or may be therapeutic in terms of a partial or complete curefor a disease and/or adverse effect attributable to the disease.“Treatment” as used herein include any treatment of a disease in amammal, and includes: (a) preventing the disease from occurring in asubject which may be predisposed to the disease but has not yet beendiagnosed as having it; (b) inhibiting the disease, i.e., arresting itsdevelopment; or (c) relieving the disease, i.e., causing regression ofthe disease. Candidate agents of interest as therapeutics forhematopoietic cancers include those that may be administered before,during or after the onset of cancer. The treatment of ongoing disease,where the treatment stabilizes or reduces the undesirable clinicalsymptoms of the patient, is of particular interest. The terms“individual,” “subject,” “host,” and “patient,” are used interchangeablyherein and include any mammalian subject for whom diagnosis, treatment,or therapy is desired, particularly humans.

As another example, engrafted genetically modified animals of thepresent disclosure are useful for studying human pathogens, i.e.pathogens that infect humans; the response of the human immune system toinfection by human pathogens; and the effectiveness of agents inprotecting against and/or treating infection by human pathogens. Thepathogen may be a virus, a fungus, a bacterium, etc. Non-limitingexamples of viral pathogens include human or porcine or avian influenzavirus. Non-limiting examples of bacterial pathogens includemycobacterium, e.g. Mycobacterium tuberculosis (M. tuberculosis), andenterobacterium, e.g. Salmonella typhi (S. typhi). Examples of methodsfor infecting mice with S. typhi and for assessing infection may befound in, for example, US Published Application No. 2011/0200982, thedisclosure of which is incorporated herein by reference. Examples ofmethods for infecting mice with M. tuberculosis and for assessinginfection may be found in, for example, US Published Application No.2011/0200982, the disclosure of which is incorporated herein byreference. Other examples of human pathogens that do not infectwild-type mice, or that infect wild-type mice but the infected mice donot model an immune response that a human mounts in response to thepathogen, will be well-known to the ordinarily skilled artisan. Suchmouse models of pathogen infection are useful in research, e.g. tobetter understand the progression of human infection. Such mouse modelsof infection are also useful in drug discovery, e.g. to identifycandidate agents that protect against or treat infection.

Engrafted genetically modified mice of the present disclosure alsoprovide a useful system for screening candidate agents for desiredactivities in vivo, for example, to identify agents that are able tomodulate (i.e., promote or suppress) hematopoietic cell developmentand/or activity, e.g. the activity of B cells, T cells, NK cells,macrophages, neutrophils, eosinophils, basophils, etc., e.g. in ahealthy or a diseased state, e.g. as cancerous cells, during pathogeninfection, for example to identify novel therapeutics and/or develop abetter understanding of the molecular basis of the development andfunction of the immune system; for agents that are toxic tohematopoietic cells, e.g. B cells, T cells, NK cells, macrophages,neutrophils, eosinophils, basophils, etc., and progenitors thereof; andfor agents that prevent against, mitigate, or reverse the toxic effectsof toxic agents on hematopoietic cells, e.g. B cells, T cells, NK cells,macrophages, neutrophils, eosinophils, basophils, etc., and progenitorsthereof; etc. As yet another example, engrafted genetically modifiedanimals of the present disclosure provide a useful system for predictingthe responsiveness of an individual to a disease therapy, e.g. byproviding an in vivo platform for screening the responsiveness of anindividual's immune system to an agent, e.g. a therapeutic agent, topredict the responsiveness of an individual to that agent.

In screening assays for biologically active agents, a humanhematopoietic cell-engrafted genetically modified mouse of the presentdisclosure, e.g. an engrafted Rag2^(−/−) IL2rg^(−/−IL-)6^(h/h) hSIRPa⁺mouse, an engrafted Rag2^(−/−)IL2rg^(−/−) IL-6^(h/h) M-CSF^(h/h)IL-3^(h/h) GM-CSF^(h/h) TPO^(h/h), hSIRPa+ mouse, etc. is contacted witha candidate agent of interest and the effect of the candidate agent isassessed by monitoring one or more output parameters. These outputparameters may be reflective of the viability of the cells, e.g. thetotal number of hematopoietic cells or the number of cells of aparticular hematopoietic cell type, or of the apoptotic state of thecells, e.g. the amount of DNA fragmentation, the amount of cellblebbing, the amount of phosphatidylserine on the cell surface, and thelike by methods that are well known in the art. Alternatively oradditionally, the output parameters may be reflective of thedifferentiation capacity of the cells, e.g. the proportions ofdifferentiated cells and differentiated cell types. Alternatively oradditionally, the output parameters may be reflective of the function ofthe cells, e.g. the cytokines and chemokines produced by the cells, theantibodies (e.g. amount or type) produced by the cells, the ability ofthe cells to home to and extravasate to a site of challenge, the abilityof the cells to modulate, i.e. promote or suppress, the activity ofother cells in vitro or in vivo, etc. Other output parameters may bereflective of the extent of damage induced by diseased hematopoieticcells, e.g. bone destruction and resorption induced by multiple myeloidcells. Yet other parameters may be reflective of the effect of the agenton infection, e.g. pathogen infection in the animal, e.g. the titer ofpathogen in the mouse, the presence of granuloma in the mouse, etc., asrelevant to the studies being performed.

Parameters are quantifiable components of cells, particularly componentsthat can be accurately measured, desirably in a high throughput system.A parameter can be any cell component or cell product including cellsurface determinant, receptor, protein or conformational orposttranslational modification thereof, lipid, carbohydrate, organic orinorganic molecule, nucleic acid, e.g. mRNA, DNA, etc. or a portionderived from such a cell component or combinations thereof. While mostparameters will provide a quantitative readout, in some instances asemi-quantitative or qualitative result will be acceptable. Readouts mayinclude a single determined value, or may include mean, median value orthe variance, etc. Characteristically a range of parameter readoutvalues will be obtained for each parameter from a multiplicity of thesame assays. Variability is expected and a range of values for each ofthe set of test parameters will be obtained using standard statisticalmethods with a common statistical method used to provide single values.

Candidate agents of interest for screening include known and unknowncompounds that encompass numerous chemical classes, primarily organicmolecules, which may include organometallic molecules, inorganicmolecules, genetic sequences, vaccines, antibiotics or other agentssuspected of having antibiotic properties, peptides, polypeptides,antibodies, agents that have been approved pharmaceutical for use in ahuman, etc. An important aspect of the invention is to evaluatecandidate drugs, including toxicity testing; and the like.

Candidate agents include organic molecules comprising functional groupsnecessary for structural interactions, particularly hydrogen bonding,and typically include at least an amine, carbonyl, hydroxyl or carboxylgroup, frequently at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules, including peptides, polynucleotides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof. Included arepharmacologically active drugs, genetically active molecules, etc.Compounds of interest include chemotherapeutic agents, hormones orhormone antagonists, etc. Exemplary of pharmaceutical agents suitablefor this invention are those described in, “The Pharmacological Basis ofTherapeutics,” Goodman and Gilman, McGraw-Hill, New York, N.Y., (1996),Ninth edition. Also included are toxins, and biological and chemicalwarfare agents, for example see Somani, S. M. (Ed.), “Chemical WarfareAgents,” Academic Press, New York, 1992).

Candidate agents of interest for screening also include nucleic acids,for example, nucleic acids that encode siRNA, shRNA, antisensemolecules, or miRNA, or nucleic acids that encode polypeptides. Manyvectors useful for transferring nucleic acids into target cells areavailable. The vectors may be maintained episomally, e.g. as plasmids,minicircle DNAs, virus-derived vectors such cytomegalovirus, adenovirus,etc., or they may be integrated into the target cell genome, throughhomologous recombination or random integration, e.g. retrovirus derivedvectors such as MMLV, HIV-1, ALV, etc. Vectors may be provided directlyto the subject cells. In other words, the pluripotent cells arecontacted with vectors comprising the nucleic acid of interest such thatthe vectors are taken up by the cells.

Methods for contacting cells, e.g. cells in culture or cells in a mouse,with nucleic acid vectors, such as electroporation, calcium chloridetransfection, and lipofection, are well known in the art. Alternatively,the nucleic acid of interest may be provided to the cells via a virus.In other words, the cells are contacted with viral particles comprisingthe nucleic acid of interest. Retroviruses, for example, lentiviruses,are particularly suitable to the method of the invention. Commonly usedretroviral vectors are “defective”, i.e. unable to produce viralproteins required for productive infection. Rather, replication of thevector requires growth in a packaging cell line. To generate viralparticles comprising nucleic acids of interest, the retroviral nucleicacids comprising the nucleic acid are packaged into viral capsids by apackaging cell line. Different packaging cell lines provide a differentenvelope protein to be incorporated into the capsid, this envelopeprotein determining the specificity of the viral particle for the cells.Envelope proteins are of at least three types, ecotropic, amphotropicand xenotropic. Retroviruses packaged with ecotropic envelope protein,e.g. MMLV, are capable of infecting most murine and rat cell types, andare generated by using ecotropic packaging cell lines such as BOSC23(Pear et al. (1993) P.N.A.S. 90:8392-8396). Retroviruses bearingamphotropic envelope protein, e.g. 4070A (Danos et al, supra.), arecapable of infecting most mammalian cell types, including human, dog andmouse, and are generated by using amphotropic packaging cell lines suchas PA12 (Miller et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Milleret al. (1986) Mol. Cell. Biol. 6:2895-2902); GRIP (Danos et al. (1988)PNAS 85:6460-6464). Retroviruses packaged with xenotropic envelopeprotein, e.g. AKR env, are capable of infecting most mammalian celltypes, except murine cells. The appropriate packaging cell line may beused to ensure that the cells of interest—in some instance, theengrafted cells, in some instance, the cells of the host, i.e. thegenetically modified animal—are targeted by the packaged viralparticles.

Vectors used for providing nucleic acid of interest to the subject cellswill typically comprise suitable promoters for driving the expression,that is, transcriptional activation, of the nucleic acid of interest.This may include ubiquitously acting promoters, for example, theCMV-β-actin promoter, or inducible promoters, such as promoters that areactive in particular cell populations or that respond to the presence ofdrugs such as tetracycline. By transcriptional activation, it isintended that transcription will be increased above basal levels in thetarget cell by at least about 10 fold, by at least about 100 fold, moreusually by at least about 1000 fold. In addition, vectors used forproviding reprogramming factors to the subject cells may include genesthat must later be removed, e.g. using a recombinase system such asCre/Lox, or the cells that express them destroyed, e.g. by includinggenes that allow selective toxicity such as herpesvirus TK, bcl-xs, etc

Candidate agents of interest for screening also include polypeptides.Such polypeptides may optionally be fused to a polypeptide domain thatincreases solubility of the product. The domain may be linked to thepolypeptide through a defined protease cleavage site, e.g. a TEVsequence, which is cleaved by TEV protease. The linker may also includeone or more flexible sequences, e.g. from 1 to 10 glycine residues. Insome embodiments, the cleavage of the fusion protein is performed in abuffer that maintains solubility of the product, e.g. in the presence offrom 0.5 to 2 M urea, in the presence of polypeptides and/orpolynucleotides that increase solubility, and the like. Domains ofinterest include endosomolytic domains, e.g. influenza HA domain; andother polypeptides that aid in production, e.g. IF2 domain, GST domain,GRPE domain, and the like. Additionally or alternatively, suchpolypeptides may be formulated for improved stability. For example, thepeptides may be PEGylated, where the polyethyleneoxy group provides forenhanced lifetime in the blood stream. The polypeptide may be fused toanother polypeptide to provide for added functionality, e.g. to increasethe in vivo stability. Generally such fusion partners are a stableplasma protein, which may, for example, extend the in vivo plasmahalf-life of the polypeptide when present as a fusion, in particularwherein such a stable plasma protein is an immunoglobulin constantdomain. In most cases where the stable plasma protein is normally foundin a multimeric form, e.g., immunoglobulins or lipoproteins, in whichthe same or different polypeptide chains are normally disulfide and/ornoncovalently bound to form an assembled multichain polypeptide, thefusions herein containing the polypeptide also will be produced andemployed as a multimer having substantially the same structure as thestable plasma protein precursor. These multimers will be homogeneouswith respect to the polypeptide agent they comprise, or they may containmore than one polypeptide agent.

The candidate polypeptide agent may be produced from eukaryotic cells,or may be produced by prokaryotic cells. It may be further processed byunfolding, e.g. heat denaturation, DTT reduction, etc. and may befurther refolded, using methods known in the art. Modifications ofinterest that do not alter primary sequence include chemicalderivatization of polypeptides, e.g., acylation, acetylation,carboxylation, amidation, etc. Also included are modifications ofglycosylation, e.g. those made by modifying the glycosylation patternsof a polypeptide during its synthesis and processing or in furtherprocessing steps; e.g. by exposing the polypeptide to enzymes whichaffect glycosylation, such as mammalian glycosylating or deglycosylatingenzymes. Also embraced are sequences that have phosphorylated amino acidresidues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine. Thepolypeptides may have been modified using ordinary molecular biologicaltechniques and synthetic chemistry so as to improve their resistance toproteolytic degradation or to optimize solubility properties or torender them more suitable as a therapeutic agent. Analogs of suchpolypeptides include those containing residues other than naturallyoccurring L-amino acids, e.g. D-amino acids or non-naturally occurringsynthetic amino acids. D-amino acids may be substituted for some or allof the amino acid residues.

The candidate polypeptide agent may be prepared by in vitro synthesis,using conventional methods as known in the art. Various commercialsynthetic apparatuses are available, for example, automated synthesizersby Applied Biosystems, Inc., Beckman, etc. By using synthesizers,naturally occurring amino acids may be substituted with unnatural aminoacids. The particular sequence and the manner of preparation will bedetermined by convenience, economics, purity required, and the like.Alternatively, the candidate polypeptide agent may be isolated andpurified in accordance with conventional methods of recombinantsynthesis. A lysate may be prepared of the expression host and thelysate purified using HPLC, exclusion chromatography, gelelectrophoresis, affinity chromatography, or other purificationtechnique. For the most part, the compositions which are used willcomprise at least 20% by weight of the desired product, more usually atleast about 75% by weight, preferably at least about 95% by weight, andfor therapeutic purposes, usually at least about 99.5% by weight, inrelation to contaminants related to the method of preparation of theproduct and its purification. Usually, the percentages will be basedupon total protein.

In some cases, the candidate polypeptide agents to be screened areantibodies. The term “antibody” or “antibody moiety” is intended toinclude any polypeptide chain-containing molecular structure with aspecific shape that fits to and recognizes an epitope, where one or morenon-covalent binding interactions stabilize the complex between themolecular structure and the epitope. The specific or selective fit of agiven structure and its specific epitope is sometimes referred to as a“lock and key” fit. The archetypal antibody molecule is theimmunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE,IgD, etc., from all sources, e.g. human, rodent, rabbit, cow, sheep,pig, dog, other mammal, chicken, other avians, etc., are considered tobe “antibodies.” Antibodies utilized in the present invention may beeither polyclonal antibodies or monoclonal antibodies. Antibodies aretypically provided in the media in which the cells are cultured.Antibody production and screen is discussed in greater detail below.

Candidate agents may be obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds, including biomolecules, includingexpression of randomized oligonucleotides and oligopeptides.Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts are available or readily produced.Additionally, natural or synthetically produced libraries and compoundsare readily modified through conventional chemical, physical andbiochemical means, and may be used to produce combinatorial libraries.Known pharmacological agents may be subjected to directed or randomchemical modifications, such as acylation, alkylation, esterification,amidification, etc. to produce structural analogs.

Candidate agents are screened for biological activity by administeringthe agent to at least one and usually a plurality of samples, sometimesin conjunction with samples lacking the agent. The change in parametersin response to the agent is measured, and the result evaluated bycomparison to reference cultures, e.g. in the presence and absence ofthe agent, obtained with other agents, etc. In instances in which ascreen is being performed to identify candidate agents that willprevent, mitigate or reverse the effects of a toxic agent, the screen istypically performed in the presence of the toxic agent, where the toxicagent is added at the time most appropriate to the results to bedetermined. For example, in cases in which the protective/preventativeability of the candidate agent is tested, the candidate agent may beadded before the toxic agent, simultaneously with the candidate agent,or subsequent to treatment with the candidate agent. As another example,in cases in which the ability of the candidate agent to reverse theeffects of a toxic agent is tested, the candidate agent may be addedsubsequent to treatment with the candidate agent. As mentioned above, insome instances, the “sample” is a genetically modified non-human animalthat has been engrafted with cells, e.g. the candidate agent is providedto an immunodeficient animal, e.g. mouse, comprising a nucleic acidencoding human IL-6 operably linked to an IL-6 promoter that has beenengrafted with human hematopoietic cells. In some instances, the sampleis the human hematopoietic cells to be engrafted, i.e. the candidateagent is provided to cells prior to engraftment into the immunodeficientgenetically modified animal.

If the candidate agent is to be administered directly to the engraftedgenetically modified animal, the agent may be administered by any of anumber of well-known methods in the art for the administration ofpeptides, small molecules and nucleic acids to mice. For example, theagent may be administered orally, mucosally, topically, intrdermally, orby injection, e.g. intraperitoneal, subcutaneous, intramuscular,intravenous, or intracranial injection, and the like. The agent may beadministered in a buffer, or it may be incorporated into any of avariety of formulations, e.g. by combination with appropriatepharmaceutically acceptable vehicle. “Pharmaceutically acceptablevehicles” may be vehicles approved by a regulatory agency of the Federalor a state government or listed in the U.S. Pharmacopeia or othergenerally recognized pharmacopeia for use in mammals, such as humans.The term “vehicle” refers to a diluent, adjuvant, excipient, or carrierwith which a compound of the invention is formulated for administrationto a mammal. Such pharmaceutical vehicles can be lipids, e.g. liposomes,e.g. liposome dendrimers; liquids, such as water and oils, includingthose of petroleum, animal, vegetable or synthetic origin, such aspeanut oil, soybean oil, mineral oil, sesame oil and the like, saline;gum acacia, gelatin, starch paste, talc, keratin, colloidal silica,urea, and the like. In addition, auxiliary, stabilizing, thickening,lubricating and coloring agents may be used. Pharmaceutical compositionsmay be formulated into preparations in solid, semi-solid, liquid orgaseous forms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres, andaerosols. The agent may be systemic after administration or may belocalized by the use of regional administration, intramuraladministration, or use of an implant that acts to retain the active doseat the site of implantation. The active agent may be formulated forimmediate activity or it may be formulated for sustained release. Forsome conditions, particularly central nervous system conditions, it maybe necessary to formulate agents to cross the blood-brain barrier (BBB).One strategy for drug delivery through the blood-brain barrier (BBB)entails disruption of the BBB, either by osmotic means such as mannitolor leukotrienes, or biochemically by the use of vasoactive substancessuch as bradykinin. A BBB disrupting agent can be co-administered withthe agent when the compositions are administered by intravascularinjection. Other strategies to go through the BBB may entail the use ofendogenous transport systems, including Caveolin-1 mediatedtranscytosis, carrier-mediated transporters such as glucose and aminoacid carriers, receptor-mediated transcytosis for insulin ortransferrin, and active efflux transporters such as p-glycoprotein.Active transport moieties may also be conjugated to the therapeuticcompounds for use in the invention to facilitate transport across theendothelial wall of the blood vessel. Alternatively, drug delivery ofagents behind the BBB may be by local delivery, for example byintrathecal delivery, e.g. through an Ommaya reservoir (see e.g. U.S.Pat. Nos. 5,222,982 and 5,385,582, incorporated herein by reference); bybolus injection, e.g. by a syringe, e.g. intravitreally orintracranially; by continuous infusion, e.g. by cannulation, e.g. withconvection (see e.g. US Application No. 20070254842, incorporated hereby reference); or by implanting a device upon which the agent has beenreversably affixed (see e.g. US Application Nos. 20080081064 and20090196903, incorporated herein by reference).

If the agent(s) are provided to cells prior to engraftment, the agentsare conveniently added in solution, or readily soluble form, to themedium of cells in culture. The agents may be added in a flow-throughsystem, as a stream, intermittent or continuous, or alternatively,adding a bolus of the compound, singly or incrementally, to an otherwisestatic solution. In a flow-through system, two fluids are used, whereone is a physiologically neutral solution, and the other is the samesolution with the test compound added. The first fluid is passed overthe cells, followed by the second. In a single solution method, a bolusof the test compound is added to the volume of medium surrounding thecells. The overall concentrations of the components of the culturemedium should not change significantly with the addition of the bolus,or between the two solutions in a flow through method.

A plurality of assays may be run in parallel with different agentconcentrations to obtain a differential response to the variousconcentrations. As known in the art, determining the effectiveconcentration of an agent typically uses a range of concentrationsresulting from 1:10, or other log scale, dilutions. The concentrationsmay be further refined with a second series of dilutions, if necessary.Typically, one of these concentrations serves as a negative control,i.e. at zero concentration or below the level of detection of the agentor at or below the concentration of agent that does not give adetectable change in the phenotype.

An analysis of the response of cells in the engrafted geneticallymodified animal to the candidate agent may be performed at any timefollowing treatment with the agent. For example, the cells may beanalyzed 1, 2, or 3 days, sometimes 4, 5, or 6 days, sometimes 8, 9, or10 days, sometimes 14 days, sometimes 21 days, sometimes 28 days,sometimes 1 month or more after contact with the candidate agent, e.g. 2months, 4 months, 6 months or more. In some embodiments, the analysiscomprises analysis at multiple time points. The selection of the timepoint(s) for analysis will be based upon the type of analysis to beperformed, as will be readily understood by the ordinarily skilledartisan.

The analysis may comprise measuring any of the parameters describedherein or known in the art for measuring cell viability, cellproliferation, cell identity, cell morphology, and cell function,particularly as they may pertain to cells of the immune cells. Forexample, flow cytometry may be used to determine the total number ofhematopoietic cells or the number of cells of a particular hematopoieticcell type. Histochemistry or immunohistochemistry may be performed todetermine the apoptotic state of the cells, e.g. terminaldeoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to measureDNA fragmentation, or immunohistochemistry to detect Annexin V bindingto phosphatidylserine on the cell surface. Flow cytometry may also beemployed to assess the proportions of differentiated cells anddifferentiated cell types, e.g. to determine the ability ofhematopoietic cells to differentiate in the presence of agent. ELISAs,Westerns, and Northern blots may be performed to determine the levels ofcytokines, chemokines, immunoglobulins, etc. expressed in the engraftedgenetically modified mice, e.g. to assess the function of the engraftedcells, to assess the survival of cancerous plasma cells, etc. μCT scansmay be performed to determine the extent of damage induced by diseasedhematopoietic cells, e.g. bone destruction and resorption induced bymultiple myeloid cells. In vivo assays to test the function of immunecells, as well as assays relevant to particular diseases or disorders ofinterest such as diabetes, autoimmune disease, graft v. host disease,AMD, etc. may also be performed. See, e.g. Current Protocols inImmunology (Richard Coico, ed. John Wiley & Sons, Inc. 2012) andImmunology Methods Manual (I. Lefkovits ed., Academic Press 1997), thedisclosures of which are incorporated herein by reference.

So, for example, a method is provided for determining the effect of anagent on multiple myeloma, comprising administering the agent to ahumanized IL-6 mouse, e.g. a Rag2^(−/−)IL2rg^(−/−)IL-6^(h/h) mouse, thathas been engrafted with human multiple myeloma cells; measuring aparameter of the viability and/or proliferative ability of the multiplemyeloma cells over time in the presence of the agent; and comparing thatmeasurement to the measurement from an engrafted humanized IL-6 mousenot exposed to the agent. The agent is determined to be anti-cancerousif it reduces the proliferation of and/or reduces the number of multiplemyeloma cells in blood or a tissue of the mouse by at least 20%, 30%,40% or more, in some instances 50%, 60%, 70% or more, e.g. 80%, 90% or100%, i.e., to undetectable amounts, following a single administrationor two or more administrations of the agent over a selected period oftime. In a specific embodiment, the administration of the drug orcombination of drugs is at least a week, 10 days, two week, three weeks,or four weeks after engraftment of the multiple myeloma cells.

Other examples of uses for the subject mice are provided elsewhereherein. Additional applications of the genetically modified andengrafted mice described in this disclosure will be apparent to thoseskilled in the art upon reading this disclosure.

Human Antibody Production

Also provided are compositions and methods useful for the production ofhuman monoclonal antibodies from an engrafted immunodeficient animal, aselsewhere described herein. In various embodiments, the methods comprisecontacting an immunodeficient animal with a human hematopoietic cell togenerate an immune system-transplanted non-human animal (engraftedanimal), subsequently contacting the engrafted animal with an antigen,collecting from the engrafted animal a human cell producing a humanantibody against the antigen, and isolating the antibody from theantibody producing cell.

In various embodiments, the invention comprises a method that includesestablishing an antibody producing cell (e.g., a human B-cell) by atransformation method (e.g. EBV) or a cell fusion method (e.g.hybridoma). Preferably the antibody producing cell is capable of beingmaintained under suitable cell culture conditions for at least about 50passages.

In various embodiments, the engrafted animal is a non-human mammal. Insome embodiments, the engrafted animal is a mouse, rat or a rabbit.

In various embodiments of the invention, the human hematopoietic cell isCD34+ cell obtained from a human fetal liver, bone marrow, cord blood,peripheral blood, or spleen sample.

In various embodiments, the antigen is at least one of: a peptide, apolypeptide, an MHC/peptide complex, DNA, a live virus, a dead virus orportion thereof, a live bacteria, a dead bacteria or portion thereof, ora cancer cell or portion thereof.

In some embodiments, the engrafted animal has been contacted with theantigen 1-5 months after the animal has been contacted with the humanhematopoietic cell. In some embodiments, the engrafted animal iscontacted only one time with the antigen, while in other embodiments,the engrafted animal is contacted two, three, four, five, six, seven,eight, or more times with the antigen.

In one embodiment, human antibody producing cell collected from theengrafted animal is a B cell. In various embodiments, the human antibodyproducing cell collected from the animal expresses on its surface atleast one of: CD19, CD20, CD22, and CD27. The human antibody-producingcell of the invention can be recovered by removal of any suitablecellular components of the immune system from the animal. In variousembodiments, the antibody-producing cell is removed from the engraftedanimal by removal of at least one of the spleen, the lymph nodes, theperipheral blood, the bone marrow or portions thereof.

In various embodiments, the method of the invention employs aconventional hybridoma technology using a suitable fusion partner. Invarious embodiments, the fusion partner is at least one cell selectedfrom the group consisting of: MOPC21, P3X63AG8, SP2/0, NS-1,P3.X63AG8.653, FO, S194/5.XXO.BU-1, FOX-NY, SP2/0-Ag14, MEG-01, HEL,UT-7, M07e, MEG-A2, and DAMI, and cell lines derived from these cells.

Methods of isolating an antibody from the engrafted animal of theinvention are well known in the art. Isolation of the antibody from theantibody producing cell, the media in which the antibody producing cellis culture, and/or the ascites of the engrafted animal, can be performedaccording to the methods known in the art, such as, by way of example,chromatography and dialysis. In other various embodiments, the antibodycan be isolated using one or more of immunoaffinity purification,ammonium sulphate precipitation, protein A/G purification, ion exchangechromatography and gel filtration. Such methods are described in Nau(1989, Optimization of monoclonal antibody purification, In: Techniquesin Protein Chemistry, Hugli, T. (ed.), Academic Press, New York) andColigan et al. (2005, Current Protocols in Immunology, John Wiley &Sons, Inc.).

The antigen may be administered to the engrafted animal by any suitablemeans known in art. In various embodiments, the antigen can beadministered to the engrafted animal by at least one ofintrasplenically, intravenously, intraperitoneally, intradermally,intramuscularly, and subcutaneously. In some embodiments, the antigen isadministered alone and in other embodiments, the antigen is administeredin combination with appropriate immunomodulating agent or adjuvant.Examples of adjuvants useful in the methods of the invention include,but are not limited to, Complete Freund's Adjuvant (CFA), IncompleteFreund's Adjuvant (IFA), and Alum (Al₃(OH)₄).

Reagents and Kits

Also provided are reagents and kits thereof for practicing one or moreof the above-described methods. The subject reagents and kits thereofmay vary greatly. In some embodiments, the reagents or kits willcomprise one or more reagents for use in the generation and/ormaintenance of the subject genetically modified non-human animals. Forexample, the kit may comprise an immunodeficient mouse comprising anucleic acid encoding human IL-6 operably linked to an IL-6 promoter anda nucleic acid encoding human SIRPa operably linked to a SIRPa promoter;or a mouse comprising a nucleic acid encoding human IL-6 operably linkedto an IL-6 promoter and further comprising a nucleic acid encoding humanM-CSF operably linked to an M-CSF promoter; a nucleic acid encodinghuman IL-3 operably linked to an IL-3 promoter; a nucleic acid encodinghuman GM-CSF operably linked to a GM-CSF promoter; a nucleic acidencoding human TPO operably linked to a TPO promoter; and/or a nucleicacid encoding human SIRPa operably linked to a SIRPa promoter. The kitmay comprise reagents for breeding such mice, e.g. primers forgenotyping for the human IL-6 gene, for the human M-CSF gene, for thehuman IL-3 gene, for the human GM-CSF gene, for the human SIRPa gene,and/or for the human TPO gene, PCR buffer, MgCl₂ solution, etc.

In some embodiments, the reagents or kits will comprise one or morereagents for use in engrafting the subject genetically modifiednon-human animals, for example human hematopoietic cells, an enrichedpopulation of human hematopoietic progenitor cells, a hematopoietic cellline, a neoplastic hematopoietic cell line, etc. for transplantationinto the subject genetically modified non-human animals, or reagents forpreparing a population of hematopoietic cells, an enriched population ofhematopoietic cells from a human, a hematopoietic cell line, aneoplastic hematopoietic cell line, etc. for transplantation into asubject genetically modified non-human animals.

In some embodiments, the reagents or kits will include reagents fordetermining the viability and/or function of hematopoietic cells, e.g.in the presence/absence of a candidate agent, e.g. one or moreantibodies that are specific for markers expressed by different types ofhematopoietic cells, or reagents for detecting particular cytokines,chemokine, etc. Other reagents may include culture media, culturesupplements, matrix compositions, and the like.

In addition to the above components, the subject kits will furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, etc., on which the information has been recorded.Yet another means that may be present is a website address which may beused via the internet to access the information at a removed site. Anyconvenient means may be present in the kits.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Example 1 Genetic Humanization of Cytokine Genes Enables Engraftment ofMice with Human Multiple Myeloma Cells

The data described herein demonstrate that the genetically modifiednon-human animals described herein represent a novel in vivo animalmodel for multiple myeloma.

Materials and Methods

Mice. Humanized IL-6 knock-in mice were generated by replacing 6.8 kb ofthe murine IL-6 gene locus with a 4.8-kb human IL-6 gene sequencecontaining exons 1 through 5 including 3′ untranslated region of thehuman IL-6 gene.

Briefly, a targeting construct for replacing the mouse with the humanIL-6 gene in a single targeting step was constructed using VELOCIGENE®genetic engineering technology (see, Valenzuela et al. (2003)High-throughput engineering of the mouse genome coupled withhigh-resolution expression analysis, Nature Biotech, 21(6):652-659).Mouse and human IL-6 DNA were obtained from bacterial artificialchromosome (BAC) RPCI-23 clone 368C3, and from BAC CTD clone 2369M23,respectively. Briefly, a NotI linearized targeting construct generatedby gap repair cloning containing mouse IL-6 upstream and downstreamhomology arms flanking a 4.8 kb human IL-6 sequence extending from ATGin exon 1 to exon 5 with 16 nucleotides of 3′ downstream sequence(genomic coordinates: NCBIh37.1: ch7:22,766,882 to 22,771,637) and afoxed neo selection cassette, was electroporated into Rag2^(+/−) IL2rg^(Y/−)ES cells. The parental ES cell line in which the RAG2 gene andIL2rg gene knockout was made was a commercially available V17 ES cell(BALB/cx129 heterozygote). Correctly targeted ES cells may beelectroporated with a transient Cre-expressing vector to remove the drugselection cassette.

Correctly targeted ES cell clones were identified by aloss-of-native-allele (LONA) assay (Valenzuela et al. 2003) in which thenumber of copies of the native, unmodified Il6 gene were determined bytwo TaqMan™ quantitative polymerase chain reactions (qPCRs) specific forsequences in the mouse 116 gene that were targeted for deletion. TheqPCR assays comprised the following primer-probe sets (written 5′ to3′): upstream forward primer, TTGCCGGTTT TCCCTTTTCT C (SEQ ID NO:1);upstream reverse primer, AGGGAAGGCC GTGGTTGTC (SEQ ID NO:2); upstreamprobe, FAM-CCAGCATCAG TCCCAAGAAG GCAACT-BHQ (SEQ ID NO:3); downstreamforward primer, TCAGAGTGTG GGCGAACAAA G (SEQ ID NO:4); downstreamreverse primer, GTGGCAAAAG CAGCCTTAGC (SEQ ID NO:5); downstream probe,FAM-TCATTCCAGG CCCTTCTTAT TGCATCTG-BHQ (SEQ ID NO:6); in which FAMrefers to the 5-carboxyfluorescein fluorescent probe and BHQ refers tothe fluorescence quencher of the black hole quencher type (BiosearchTechnologies). DNA purified from ES cell clones that have taken up thetargeting vector and incorporated in their genomes was combined withTaqMan™ Gene Expression Master Mix (Life Technologies) according to themanufacturer's suggestions in a 384-well PCR plate (MicroAmp™ Optical384-Well Reaction Plate, Life Technologies) and cycled in an AppliedBiosystems Prism 7900HT, which collects fluorescence data during thecourse of the PCRs and determines a threshold cycle (Ct), the fractionalPCR cycle at which the accumulated fluorescence reaches a pre-setthreshold. The upstream and downstream Il6-specific qPCRs and two qPCRsfor non-targeted reference genes were run for each DNA sample. Thedifferences in the Ct values (ΔCt) between each Il6-specific qPCR andeach reference gene qPCR were calculated, and then the differencebetween each ΔCt and the median ΔCt for all samples assayed wascalculated to obtain ΔΔCt values for each sample. The copy number of theIL-6 gene in each sample was calculated from the following formula: copynumber=2·2^(−ΔΔCt). A correctly targeted clone, having lost one of itsnative copies, will have an IL-6 gene copy number equal to one.Confirmation that the human IL-6 gene sequence replaced the deletedmouse Il-6 gene sequence in the humanized allele was confirmed by aTaqMan™ qPCR assay that comprises the following primer-probe sets(written 5′ to 3′): the human forward primer, CCCCACTCCACTGGAATTTG (SEQID NO:7); the human reverse primer, GTTCAACCACAGCCAGGAAAG (SEQ ID NO:8);and the human probe, FAM-AGCTACAACTCATTGGCATCCTGGCAA-BHQ (SEQ ID NO:9).

The upstream junction of the murine locus and the sequence containingthe hIL-6 gene is designed to be within 5′-AATTAGAGAG TTGACTCCTAATAAATATGA GACTGGGGAT GTCTGTAGCT CATTCTGCTC TGGAGCCCAC CAAGAACGATAGTCAATTCC AGAAACCGCT ATGAACTCCT TCTCCACAAG TAAGTGCAGG AAATCCTTAGCCCTGGAACT GCCAGCGGCG GTCGAGCCCT GTGTGAGGGA GGGGTGTGTG GCCCAGG (SEQ IDNO:10), wherein the final mouse nucleotide prior to the first nucleotideof the human gene is the “T” in CCGCT, and the first nucleotide of thehuman sequence is the first “A” in ATGAA. The downstream junction of thesequence containing the hIL-6 gene and the murine locus is designed tobe within 5′-TTTTAAAGAA ATATTTATAT TGTATTTATA TAATGTATAA ATGGTTTTTATACCAATAAA TGGCATTTTA AAAAATTCAG CAACTTTGAG TGTGTCACGC TCCCGGGCTCGATAACTATA ACGGTCCTAA GGTAGCGACT CGAGATAACT T-3′ (SEQ ID NO:11), whereinthe final nucleotide of the human sequence is with the final “G” inTCACG and the first nucleotide of the mouse sequence is the first “C” inCTCCC; the downstream junction region also contained a loxP site at the3′ end (the beginning of which is shown) for removal of a foxedubiquitin promoter-driven neo cassette. The junction of the neo cassettewith the mouse IL-6 locus is designed to be within 5′-TATACGAAGTTATCCTAGGT TGGAGCTCCT AAGTTACATC CAAACATCCT CCCCCAAATC AATAATTAAGCACTTTTTAT GACATGTAAA GTTAAATAAG AAGTGAAAGC TGCAGATGGT GAGTGAGA (SEQ IDNO:12), where the final “C” of AGCTC is the final nucleotide of the neocassette; the first nucleotide of the mouse genome following thecassette is the initial “C” of CTAAG.

To generate a mouse comprising hIL-6 and lacking Rag2 and Il2rg,correctly targeted ES cells are identified, and are introduced intopreimplantation embryo using techniques known in the art.

Humanized IL-6 KI mice were then backcrossed to generate mice lackingRag2 and Il2rg and expressing hIL-6, and crossed to mice expressing ahuman SIRPa transgene (Strowig et al., 2011, Proc Natl Acad Sci USA,108(32): 13218-13223) to generate mice deficient for Rag2 and Il2rg aswell as expressing both hIL-6 and hSIRPa (Rag2^(−/−)Il2rg^(null)Il6^(h/h)hSIRI³a⁺). In addition, Rag2^(−/−), IL-2rg^(Y/−), hIL-6 KI micewere crossed with mice expressing human TPO (Rongvaux et al., 2011, ProcNatl Acad Sci USA, 108(6): 2378-2383), human IL-3 and human GM-CSF(Willinger et al, 2011, Proc Natl Acad Sci USA, 108(6): 2390-2395), andhuman M-CSF (Rathinam et al, 2011, Blood, 118(11): 3119-3128) as well ashSIRPa (Strowig et al., 2011, Proc Natl Acad Sci USA, 108(32):13218-13223) to generate mice expressing a combination of these humanproteins (Rag2^(−/−) Il2rg^(null)hSIRPa⁺ Tpo^(h/h) Mcsf^(h/h)Il3/Gmcsf^(h/h) Il6^(h/h)).

Cell lines and primary cells. The multiple myeloma cell line INA-6(Burger et al., 2001, Hematol J, 2(1): 42-53) was maintained in RPMI1640medium supplemented with 20% FCS, penicillin/streptomycin, L-glutamine,and 2.5 ng/ml of hIL-6 in a standard incubator at 37 C and 5% CO₂.

Primary cells from multiple myeloma patients were isolated from bonemarrow aspirates after obtaining informed consent from patients.Mononuclear cells were purified by Ficoll density-gradientcentrifugation and subsequently, different cell subsets were isolated byMagnetic-activated cell sorting)(MACS®). To obtain, T cell-depletedpopulations, CD3+ cells were depleted by negative selection on anAutoMACS system using anti-CD3 microbeads (Miltenyi Biotec). To obtainCD138+ cells, CD138+ cells were isolate by positive selection on anAutoMACS® system using anti-CD138 microbeads (Miltenyi Biotec). Purityof cells after MACS® selection was analyzed by flow cytometry.

Transplantation of cells. For intrafemoral transplantation of INA6cells, Rag2^(−/−) Il2rg^(null) Il6^(h/h)hSIRPa⁺ mice were irradiatedtwice with 200 rad from an X-ray source. Indicated amounts of cells werethen transplanted into the femur of recipient mice. Briefly, mice wereanaesthetized using Ketamine/Xylazine and a hole was drilled into thepatellar surface of the femur using a 26 gauge needle. The cells werethen slowly injected in a volume of 20 μl using a 27 gauge needle. Fortransplantation of primary patient-derived cells,Rag2^(−/−)Il2rg^(null)hSIRPa⁺ Tpo^(h/h) Mcsf^(h/h) Il3/Gmcsf^(h/h)Il6^(h/h) mice were irradiated twice with 150 rad from an X-ray sourceand transplantation was performed as described above.

ELISA. Commercial ELISA kits were used to measure the concentrations ofhuman soluble IL-6R (R&D Systems), human Igκ, and Igλ, (BethylLaboratories). Detection of these proteins was performed according tomanufacturer's instructions.

μCT. Femur morphometry was quantified using cone-beam microfocus x-raycomputed tomography (μCT40; ScancoMedicalAG). Serial tomographic imageswere acquired, and 3D images were reconstructed and used to determinethe parameters. Trabecular morphometry was characterized by measuringthe bone volume fraction, trabecular thickness, trabecular number, andtrabecular separation. Cortical measurements included average corticalthickness, cross-sectional area of cortical bone, subperiostealcross-sectional area, and marrow area.

Histology. Femurs were stripped of soft tissue, fixed in 10% bufferedformalin, dehydrated, and embedded in methyl methacrylate before beingsectioned and stained with toluidine blue according to standardprocedures.

Results

Engraftment of multiple myeloma cell line in mice with humanized IL-6gene. A human IL-6 dependent MM cell line (INA6-gfp) was utilized toevaluate if mice expressing human SIRPα and IL-6 are suitable hosts formultiple myeloma (MM) cell lines. The INA6-gfp cell line shows highdependency on human microenvironment, i.e., human fetal bone chips, whentransplanted in xenograft systems of scid-hu mice (Epstein et al., 2005,Methods Mol Med, 113: 183-190). Specifically, INA-6 cells are only ableto engraft the human bone graft in scid-hu mice, suggesting dependenceon a human bone marrow microenvironment, similar to primary MM cells(Tassone et al., 2005, Blood, 106(2): 713-716).

Hence, to directly test the potential of IL-6 humanization in enablingthe growth of myeloma cells, INA-6 cells were transplanted intravenouslyinto i) Rag2^(−/−)Il2rg^(null), ii) Rag2^(−/−)Il2rg^(null)hSIRPa+, iii)Rag2^(−/−)Il2rg^(null) Il6^(h/h), and iv) Rag2^(−/−)Il2rg^(null)Il6^(h/h)hSIRPa+ mice. Engraftment was analyzed by measuring sIL-6Rprotein secreted by INA-6 cells in the blood. Engraftment was onlydetected in mice expressing human IL-6, (FIG. 1) demonstrating thatINA-6 cells were indeed able to engraft mice expressing human IL-6.Next, the location of INA-6 cells (modified to express GFP) in theengrafted mice was investigated by fluorescent microscopy. Few GFP+cells was detected in the bone marrow (FIG. 2), but an increased numberof GFP+ cells was detected in the lung of engrafted mice (FIG. 3).

Analysis of human 116 gene expression revealed that the highest level ofhuman 116 gene expression was found in the lung (FIG. 3), hencecorrelating with the presence of INA-6 cells in the lung. In summary,the data disclosed herein demonstrate the successful engraftment ofINA-6 cells after intravenous injection (i.v.) into mice geneticallymodified to express human IL6 or human IL6 and SIRPa, suggesting thatgenetic humanization is able to overcome growth restrictions of human MMcells in mice.

The data in FIG. 3 suggests that INA-6 cell do not home efficiently tothe bone marrow and may grow instead at non-physiological sites.Therefore, it was next examined whether transplantation of the tumorcell line in its natural microenvironment is able to reproduce thepathology typically associated with human MM. To do so, intraboneinjection of INA6 cells was tested. This strikingly resulted in bonedestruction and resorption, which are aspects of the pathology seen inMM patients (FIG. 4-6). Specifically, the loss of trabecular bone masswas observed by histology, which was quantified by μCT. Moreover, onlylimited metastases to peripheral sites, such as the lung, was observed,which leads to the conclusion that the model may be further explored toinvestigate novel drugs interfering with this pathology. To test thisconclusion INA-6-engrafted mice were treated with Zometa or Velcade, twodrugs commonly used to treat multiple myeloma patients. Strikingly,Zometa treatment of mice injected with INA-6 cells was able to reducebone resorption compared to untreated mice as quantified by μCT (FIG.7).

The data disclosed herein demonstrate that humanization of the Il6 geneenables engraftment of multiple myeloma cell lines that typicallyrequire a human microenvironment. Engraftment recapitulates severalpathological symptoms observed in patients including bone loss. Further,these symptoms can be treated using approved drugs highlighting theutility of this model to test new drugs.

Genetic humanization of cytokine genes enables engraftment of primarypatient-derived multiple myeloma cells. Next, experiments were conductedto examine the transplantation of primary MM cells into geneticallyhumanized mice. It has been previously demonstrated that humanization ofmultiple cytokines including thrombopoietin, IL-3, GM-CSF, and M-CSF aswell as the macrophage inhibitory receptor SIRPa results in improvedengraftment of human hematopoietic cells in immunodeficient mice(Strowig et al., 2011, Proc Natl Acad Sci USA, 108(32): 13218-13223;Rathinam et al, 2011, Blood, 118(11): 3119-3128; Rongvaux et al., 2011,Proc Natl Acad Sci USA, 108(6): 2378-2383; Willinger et al, 2011, ProcNatl Acad Sci USA, 108(6): 2390-2395). Specifically, humanization ofIL-3 and GM-CSF as well as M-CSF improved engraftment of myeloid cellsthat have been demonstrated to be important for specific aspects of MMpathology. Transgenic expression of hSIRPa improves human cellengraftment, but the SIRPa-CD47 axis has also been recently implicatedin tumorgenesis. Previously generated humanized mice were combined withhuman IL-6 knock-in mice to generate Rag2^(−/−)Il2rg^(null)hSIRPa⁺Tpo^(h/h) Mcsf^(h/h) Il3/Gmcsf^(h/h) Il6^(h/h) mice. To evaluate theability of this strain to support human cell engraftment, CD3-depletedbone marrow cells from MM patients was injected into the bone marrow ofthe mice. A high frequency of myeloma cells, as identified asCD138+CD38+CD19− cells, was detected in the injected bone, while onlyfew cells were detected in the collateral bone (FIG. 8).

These results suggest that the genetically humanized mice, describedherein, support the engraftment of primary human MINI cells in vivo. Thepresent data demonstrates that the humanization of cytokines geneencoding IL-6, TPO, IL-3, GM-CSF, and/or M-CSF enable engraftment ofprimary multiple myeloma cells from patients that typically require ahuman microenvironment for successful transplantation.

Example 2 Genetic Humanization of the IL-6 Gene Enables Engraftment ofMice with Human Hematopoietic Cells

Materials and Methods

Mice. The humanized IL-6 KI mouse was generated as described above. Thechimeric mice were first bred with BALB/c mice and then backcrossed inorder to obtain offspring with hIL-6 in homozygosity. Mice with the samemixed BALB/c×129 background were used as control.

Newborn pups (within first day of life) were sublethally irradiated byX-ray irradiation in a 4-hour interval with 2×150cGy. Four hours afterthe irradiation the mice were injected with 1-2×10⁵ CD34⁺ fetal liver(FL) cells in 20 μl of PBS into the liver by using a 30-gauge needle(Hamilton Company, NV, USA). Mice were weaned at 3 weeks of age. Themice were maintained under specific pathogen-free conditions and allexperiments were performed in compliance with Yale Institutional AnimalCare and Use Committee protocols.

Analysis of human and mouse hematological cell populations. The micewere bled from retro-orbital plexus under isofluorane anesthesia atdifferent times after transplantation. Plasma samples were collected andstored at −20° C. for further Ig measurement. Red blood cells were lysedtwo times by using Ammonium-Chloride (ACK) lysing buffer and theremaining PBMC were resuspended in FACS buffer (PBS supplemented with 5%FBS and 5 mM EDTA).

When the mice were killed, a single-cell suspension of cells wasobtained from the bone marrow (BM), thymus and spleen. Samples were thenstained with fluorochrome-labeled mAbs against mouse and human cellsurface antigens according to the manufactures' instructions. Thefollowing anti-human mAbs were used: CD45 (HI30), CD19 (HIB19), CD3(UCHT1), CD4 (RPA-T4), CD8 (HIT8a), CD20 (2H7), CD5 (UCHT2), CD27(O323), CD24 (ML5), CD10 (HI10a), all from Biolegend, Calif., USA; CD33(WM53), CD38 (HIT2), IgM (G20-127), CD138 (MI15) from BD Biosciences andCD34 (AC136) from Miltenyi Biotec. The mouse cells were stained with ananti-murine CD45 Ab (30-F11, Biolegend). Samples were acquired on theLSRII (BD Biosciences) cytometer and analyzed on FlowJo (Treestar,Oreg., USA) software.

Measurement of human immunoglobulins. Total immunoglobulins (Igs) weremeasured in the plasma collected from the mice by ELISA. 96 well plates(Nunc, N.Y., USA) were coated at 4° C. overnight with 20 μg/ml purifiedgoat anti-human IgM and IgG (Southern Biotechnology, AL, USA). Afterwashing and blocking with PBS 1% bovine serum albumin (BSA,Sigma-Aldrich) appropriate dilutions of the samples were added for 2hours at room temperature (RT). Plates were washed and incubated for 1hour at RT with isotype specific secondary biotinylated antibodies(Southern Biotechnology) followed by Streptavidin-HRP (Pierce ProteinResearch Products, IL, USA). After a final wash the enzyme activity wasdetermined using the TMB substrate solution followed by the stopsolution (both from Pierce Protein Research Products). The absorbancewas measured at 450 nm. A sample of human serum from Bethyl (TX, USA)was used as reference.

Statistical analysis. All data were expressed as average±standard errorof mean (SEM) with the exception of the Ab levels that were plotted asgeometric mean. The non-parametric Mann-Whitney U test was used todetermine statistical significance between two groups. Differences wereconsidered significant when the p values were lower than 0.05.

Results

Peripheral blood engraftment in human CD34⁺ cell transplanted RAG2^(−/−)γ_(c) ^(−/−) mice. The IL6 h/h mice showed a higher peripheral blood(PB) engraftment throughout the time of the analysis when compared tothe IL6 m/m mice and their engraftment increased over time (FIG. 9).There were no major differences in the composition of the human cellsbetween the 2 groups of mice at any time point tested (FIG. 10). In bothgroups B and T cell percentages were similar at week 8 and week 16-20whereas at weeks 11-15 there was a higher percentage of B cells(69.23±3.97 in IL6 m/m and 55.91±4.86 in IL6h/h) than T cells(16.86±3.14 in IL6 m/m and 30.26±6.23 in IL6h/h). Myeloid CD33⁺ cellsrepresented a minor component of the human cell and their percentagedecreased over time.

Organ cell engraftment and composition of human CD34⁺ cell reconstitutedRAG2^(−/−) γ_(c) ^(−/−) mice. Cells of human origin were found in thehemato-lymphoid organs, such as BM, spleen and thymus (FIG. 11, PanelA). Interestingly the spleen of the IL6 h/h mice displayed a greaterhuman engraftment than IL6 m/m mice (61.75%±10.87 versus 23.56%±5.2).These data were confirmed by the doubling in the absolute number ofhuman cells (14.21×10⁶±1.38 versus 7.26×10⁶±0.66) (FIG. 11, Panel B).

B cell maturation in human CD34⁻ engrafted RAG2^(−/−)γ_(c) ^(−/−) mice.The maturation stages of the human B cells were studied in the BM andspleen of the engrafted mice using the gating strategies illustrated inFIG. 12, Panel A, based on the usage of a combination of CD24, CD38 andsurface IgM antibodies. This strategy is particularly helpful indissecting the presence of the transitional B cell subset. In BM themain compartment was made by the Pro-/Pre-B cells (FIG. 12, Panel B)with no difference between the IL6 m/m and the IL6 h/h mice (76.04%±9.09and 79.07%±3.43 respectively). The spleen of both groups contained somemature B cells (˜20%) but still a high percentage ofimmature/transitional cells (27.13%±8.99 and 36.45%±5.12).

A significant percentage of B cells in the spleen were CD5⁺(FIG. 13,Panel B), a marker not commonly expressed on human BM and peripheral Bcells but found in low percentage on the FL B cells (FIG. 13, Panel A).

The IL6 h/h mice showed a sharp increase in the percentage of the CD27⁺B cells in the spleen when compared to the IL6 m/m mice (24.73%±8.94versus 9.46%±2.32) but very few CD27⁺ cells were found in the BM (FIG.14, Panels A and B).

Antibody production in human CD34⁺ engrafted RAG2^(−/−)γ_(c) ^(−/−)mice. As the B cells were found in the engrafted mice, the concentrationof the human IgM and IgG in the plasma collected at 12 and 20 weeksafter the human cell transplantation was then measured. Both IL6 m/m andIL6 h/h mice secreted human IgM and IgG (FIG. 15).

In general there was a higher percentage of IL6 h/h mice secretingantibodies compared to the IL6 m/m ones with the former mice showing asmaller level of IgM level (14.69 μg/ml versus 33.66 μg/ml at week 12and 18.25 μg/ml versus 190.2 μg/ml at week 20) but an increased level ofIgG (243 μg/ml versus 49.6 μg/ml at week 12 and 553.6 μg/ml versus 297.2μg/ml at week 20) (FIG. 15, Panel A). Both IgM and IgG average levelrose over time in IL6 m/m mice (FIG. 15, Panel B). Conversely the serumIgs remained steady in IL6 h/h mice.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

The invention claimed is:
 1. A method of generating a mouse model ofhuman B cell development and function, the method comprising:transplanting a population of human hematopoietic cells into agenetically Rag2^(−/−), IL2rg^(null) modified mouse that isimmunodeficient and comprises in its genome a nucleic acid encodinghuman IL-6 operably linked to the endogenous mouse IL-6 promoter at themouse IL-6 locus, wherein the mouse expresses human IL-6 and does notexpress mouse IL-6, and wherein the mouse produces human B cells.
 2. Themethod of claim 1, wherein the transplanted population of hematopoieticcells comprises CD34+ cells.
 3. The method of claim 1, wherein thetransplanted population of hematopoietic cells comprises multiplemyeloma cells.
 4. The method of claim 1, wherein the transplantingcomprises intrafemoral and/or intratibial injection.
 5. The method ofclaim 1, wherein the mouse further comprises in its genome a nucleicacid encoding human SIRPa, or a nucleic acid encoding a fusion proteincomprising a biologically active fragment of a full length human SIRPApolypeptide, operably linked to a SIRPa promoter.
 6. The method of claim5, wherein the SIRPa promoter is the mouse SIRPa promoter, and thenucleic acid encoding human SIRPa, or the nucleic acid encoding a fusionprotein comprising a biologically active fragment of a full length humanSIRPA polypeptide, is operably linked to the mouse SIRPa promoter at themouse SIRPa locus.
 7. The method of claim 1, wherein the mouse comprisesin its genome at least one additional nucleic acid selected from thegroup consisting of: i) a nucleic acid encoding human M-CSF operablylinked to a M-CSF promoter; ii) a nucleic acid encoding human IL-3operably linked to an IL-3 promoter; iii) a nucleic acid encoding humanGM-CSF operably linked to a GM-CSF promoter; and iv) a nucleic acidencoding human TPO operably linked to a TPO promoter.
 8. The method ofclaim 7, wherein the M-CSF promoter is the mouse M-CSF promoter, and thenucleic acid encoding human M-CSF is operably linked to the mouse M-CSFpromoter at the mouse M-CSF locus, the IL-3 promoter is the mouse IL-3promoter, and the nucleic acid encoding human IL-3 is operably linked tothe mouse IL-3 promoter at the mouse IL-3 locus, the GM-CSF promoter isthe mouse GM-CSF promoter, and the nucleic acid encoding human GM-CSF isoperably linked to the mouse GM-CSF promoter at the mouse GM-CSF locus,and the TPO promoter is the mouse TPO promoter, and the nucleic acidencoding human TPO is operably linked to the mouse TPO promoter at themouse TPO locus.
 9. The method of claim 1, wherein the transplantingcomprises injecting the population of human hematopoietic cells into theliver of the mouse.
 10. The method of claim 3, wherein the humanmultiple myeloma cells are INA6 cells.